TW200808973A - Granulysin and uses thereof - Google Patents

Abstract

Disclosed are uses of granulysin in methods of diagnosing or treating autoimmune disorders.

Description

IX. OBJECTS OF THE INVENTION: TECHNICAL FIELD The present invention relates to a method for detecting and treating diseases associated with unnecessary immune reactions using granulysin. [Prior Art] Unnecessary immune response is associated with the occurrence of many physical disorders. 'These disorders include adjive drUg reactions 'ADRs', transplants against host diseases (graf^versus_h〇Sf chsease, GVHD), inflammatory diseases, body-immune diseases, transplant rejection, allergic diseases, and cancers derived from T cells, all of which are major clinical problems. For example, adverse reactions of music The Stevens-Johnson syndrome (sjs) and t〇xic epidermal necrolysis (ten), which account for all hospitalization reasons, are characterized by a large number of fatal skin disorders with keratinocyte necrosis. The reaction, and the two diseases of the same severity have the characteristics of rapid development into purple-spotted vesicular rash and similar damage with a wide range of mucosal invasion and skin exfoliation (preciseness, · N· Engl·J · Med· 1994 细1〇;331(19): 1272 Milk syndrome is defined as the degree of skin exfoliation is less than 1%, and the degree of skin exfoliation exceeds 30%. Toxic epidermal necrolysis, and text between Steve Johnson syndrome and no epidermal necrosis skin peeling bullosa is as 10_30% (Ro_au type j · As it is Derma 1994200808973

Jun; 102 (6): 28S-30S). Histopathological observations of Steven Johnson's Johnson & Johnson syndrome and toxic epidermal necrolysis include significant keratinocyte dying in the epidermis associated with epidermal dermis separation and epidermal necrosis, resulting in blisters and large-scale skin mucosal shedding (Paul did not乂Br. J. Dermatol. I"6 Apr;134(4):710_4). In addition to severe skin symptoms, Steven Johnson's Johnson & Johnson syndrome and toxic epidermal necrolysis are associated with fever, myocardial I, inflammation, myocardial infarction, hepatitis, acute renal failure, and 1 affecting the respiratory and gastrointestinal systems. Although the incidence of Stellen's Johnson & Johnson syndrome and toxic epidermal necrolysis is not high, these conditions can cause death or severe disability in people who were previously in good health. A few cases may even prompt pharmaceutical companies to recycle newly marketed drugs. (Roujeau 汊fl/·, N. Engl. J. Med. lMf Nov l〇; 331(19): 1272-85) 〇 Therefore, a need is available for testing Or a method of treating a disease associated with an unwanted immune response. SUMMARY OF THE INVENTION The present invention relates to the detection and treatment of granulysin (granulysin) with unnecessary immune response _ disease, such as toxic T cell regulation disorder (eytotoxieTeelknediateddisOTtos) granulolysin difficult autoimmune disorder ( g_lySin_mediated auto_une disorders ) and autoimmune disorders. A lx month of the purpose of 'for the test - whether the individual has one or more secrets _ method granules, such as the lysin regulated by the 200808973 autoimmune disorder. The method comprises: obtaining a test sample of one body; determining a performance amount of the particulate lysin in the test sample; the performance amount is compared with a preset value. If the particle lysin expression amount in the test sample is higher than the preset The value 'to thereby determine that the individual has an autoimmune disorder or a risk of developing granulysin, which may be Steven's Johnson & Johnson syndrome, toxic epidermal necrolysis, transplantation versus host disease (gmft-Versus- Host disease ), Behcet disease, ankylosing sp〇ndyiitis, systemic lupus erythematosus (syste) Mic lupus erythematosus ), dermatomyositis (demiatoniyositis), polymyositis (p〇1ymy〇sitis)' and state B transplant rejection. The test sample can be a body fluid sample, such as a blister sample or a serum sample. The granulysin includes a sequence of 9 ki>a type (SEQ ID NO: 1), a sequence of 15 kDa type (SEQ ID NO: 3) or an antigen fragment of one of the sequences. The following is an amino acid sequence (SEQ ID NO: 1) And SEQ Π) NO: 3) and the nucleotide sequence encoding the polypeptide (SEQ ID NO: 2 and SEQ ID NO: 4), and SEQ ID Ν 0··3 removed the sequence of the 9 kDa type (SEQ ID NO: 1) This is the underlined part (SEQ IDN0:5). Wherein the antigenic fragment has at least one (eg, Π, 15, 18, 30, 50, or 100) amino acid residue length, for example, the antigen fragment comprises SEQ ID NO: 5 or a fragment thereof . SEQIDN01:

GRDYRTCLTIVQKLKKMVDKPTQRSVSNAATRVCRTGRSRWRDVCRNFMRRY

QSRVTQGLVAGETAQQICEDLRLCIPSTGPL 7 SEQIDN0 2: ggcGgtgactacaggacctgtctgacgatagtccaaaaaGtgaagaag atggtggdtaagcccacccagagaagtgtttccaatgctgcgacccgg gtgtgtaggacggggaggtcacgatggcgcgacgtctgcagaaatttc atgaggaggtatcagtctagagttacccagggcctcgtggccggagaa actgcccagcagatctgtgaggacctcaggttgtgtataccttctaca ggtcccctctga SEQIDN0 3:

MATWALLjLLAAMLLGNPGLVFSRLSPEYYDLARAHLRDEEKgCPCLAQ Ε6ΡΩ6ΡΙ^ΤΚΤ〇ΕΕ〇ΐωΥΚΤ(:Ι^ΤΊν(2Κ]^ΚΚΜν〇ΚΡΤ〇Ι^ν3ΝΑΆΤΚν〇

RTGRSRWRDVCRNF^RRYQSRVTQGLVAGETAQQICEDLRLCIPSTGP

L SEQ ID NO 4: atggctacctgggccctcctgctccttgcagccatgctcctgggcaac ccaggtctggtcttctctcgtctgagccctgagtactacgacctggca agagcccacctgcgtgatgaggagaaatcctgcccgtgcctggcccag gagggcccccagggtgacctgttgaccaaaacacaggagctgggccgt gactacaggacctgtctgacgatagtccaaaaactgaagaagatggtg gataagcccacccagagaagtgtttccaatgctgcgacccgggtgtgt aggacggggaggtGacgatggcgcgacgtctgcagaaatttcatgagg aggtatcagtctagagttacccagggcctcgtggccggagaaactgcc cagcagatctgtgaggacctcaggttgtgtataccttctacaggtccc ctctga the present invention is drawn up to monitor whether a Mian body having one or more of the foregoing disorders based method, the method comprising determining from a body of the test particles in the sample The amount of lysin expressed. The invention further encompasses a method of assessing a subject who is scheduled to receive or has received a medical treatment or a transplant. The method comprises determining the amount of granulysin in a test sample taken from a body, and if the granule lysin expression in the test sample of the individual is lower than a predetermined value, indicating that the individual has a good prognosis; If the amount of granulysin in the test sample is higher than the preset value, the individual is inferior in prognosis. Based on the results, the doctor can use it to determine if the individual is eligible for treatment or organ transplantation. The method goes into 200808973

The step comprises obtaining a sample of the individual before or after the 'providing-individual-treatment of the therapeutic agent (e.g., drug) or transplanting the plant (e.g., a cell or a tissue) to determine if the individual is prone to adverse drug reactions. For the same purpose, the method also includes measuring the granule solute in the sample by contacting the sample or other sample (such as a sample containing tau cells) with a therapeutic agent or a graft. the amount. The preset value can be obtained by a healthy individual or a body having one or more of the aforementioned disorders (e.g., Steven's syndrome/toxic epidermal necrolysis) according to the following method. - Grafts refer to the transplantation of organs, tissues or cells (such as stem cells) into the same region of the same individual or transplanted to another body. 0 * This 'X(10) purpose' is treated with _ treatment - or a variety of the aforementioned disorders The method of the main method comprises the administration of an effective dose of the granulysin to the individual, and an antibody, or an RNA molecule. The stalk-re-purpose is based on the determination of the compound to be tested for the treatment of multiple species or multiple deafness methods. The method consists of a fine touch of a test substance and a granule, followed by Test = the amount of particle lysis in the presence of a compound with or without a compound. If the amount of __ granulysin is lower than the presence of the compound to be tested in the presence of the substance to be tested, that is, it should not be treated as an imbalance. Another identifiable test method for treating the aforementioned disorders, comprising providing a granule-dissolving scaly touch; a peptide-and-compound compound 200808973, molecular contact; and detecting the multi-peptide and the test compound Intermolecular binding. If the test compound molecule binds to the multi-peptide, the compound is determined to be a candidate compound for the aforementioned disorder. Many granulysins and their isomers can be used in the present invention, and examples of octasol and its isomers are described in Krenskyeb/. Am. J., she. 2005, fine; 5(8): 1789-92; Atvierson de J Mol · 2003 Jan 10325 (2) 355-65; Gamen et al J. Immunol. 1998 • Aug 15,161(4)·1758,64; Pardo ei a/. J. Immunol. 2001 Aug l;167(3):1222-9; and Deng et al I Immunol 2005 May 1;174(9):5243·8. The detailed description of the specific embodiments of the present invention is set forth in the appended claims [Embodiment] • The Department of Health is based on an unanticipated discovery that granulysin involves an unnecessary immune response in the side of the disease, such as dystrophic syndrome, toxic epidermal necrosis. Lysis or transplantation against host disease. The origin of the strong biota and the poisonous Zhao Zhaozhi is not fully understood yet, but the re-reading of the drug will shorten the latent side and lead to stricter (4) recordings. These are serious fatal drugs. Caused by the regulation of immune response (test 4 10 200808973

Toxicology· 2005 Apr 15;209(2):123_9). The clinical, histopathological, immunocytological, and functional findings of Steven's Johnson & Johnson syndrome and toxic epidermal necrolysis support the following statement: Steven's Johnson & Johnson syndrome/toxic epidermal necrolysis A unique drug allergic reaction triggered by toxic lymphoblasts. The in vivo (in vitr〇 ) study showed that the drug response was defined as the first type of histocompatibility complex.

I (major histocompatibility complex, MHC) causes a large proliferation of CD8 intestinal cytotoxic T lymphocytes (CTLs) and these cells trigger a cytotoxic response. Recently, it has been found that the allele of human humen leukocyte antigen (HLA-β) has a strong gene phase and sputum relationship with specific drug reactions (see Chung β a/· Nature· 2004 Apr 1 ; 428 ( 6982): 486), this finding supports the concept of a defined histocompatibility complex of a drug or its metabolites that have led to the concept of T cell activation' and the poisonous tau cells infiltrate the Stellen Johnson syndrome and toxic epidermal necrosis Skin lesions in patients with lysis (see Nassif ei a/·, J· Allergy Clin. Immunol. 2004 Ν〇ν; 114(5)··1209_15). Tau cells in vesicles and epidermis exhibit a CD8 positive phenotype (see Nassif ei a/., J· Invest Dermatol· 2002)

Apr; 118 (4): 728_33). Based on the above findings, it is shown that the occurrence of Strychn's Johnson & Johnson syndrome and toxic epidermal necrolysis is mainly caused by antigen-based and poisonous T cells. Although it is believed that the pathogenesis of Stellen's Johnson & Johnson syndrome and toxic epidermal necrolysis is related to immune regulation, the unique risk signal that causes a large number of skin cell deaths is still unknown. 11 200808973 As mentioned, granulysin was found to be a key molecule responsible for the unique clinical manifestations of Steven's Johnson & Johnson and toxic epidermal necrolysis. For example, vesicular fluids derived from the skin lesions of Steven Johnson's Johnson & Johnson syndrome and toxic epidermal necrolysis have anti-B cell and keratinocyte cytotoxic activity, and the overall gene expression profile of vesicular cells has revealed granulysin The most important cytotoxic protein, this result was confirmed by real-time quantitative polymerase chain reaction (RT-qPCR) and immunohistochemical analysis. In vivo experiments (z>2 v/vo) found that injection of granulysin into the epidermis of mice resulted in the death of a large number of skin cells, and the episodes of epidermal necrolysis with human history ♦ dynasty Johnson syndrome and venom Similar in shape. Therefore, the key molecular response to the diffuse keratinocyte apoptosis of Stellen's Johnson & Johnson syndrome/toxic epidermal necrolysis and the cornerstone of its pathogenic mechanism are granulysin, while granzyme/perforation Perforin or soluble Fas ligand (sFasL). The special correlation between the vesicular fluid and granulysin of Steven's Johnson & Johnson syndrome/toxic epidermal necrolysis also shows that granulysin can be used for the detection of other pustular skin diseases, thus avoiding skin biopsy. In addition, granulysin can also be used as a target for new therapies to develop these lethal diseases. Method for detecting or pre-existing The scope of the present invention is a multi-peptide or a detection or a ship (four) material using granulysin. The method for assessing the granule lysin gene is included in 12 (2008), and the method can be applied. Detection or prognosis of autoimmune disorders, including autoimmune disorders regulated by granulysin. Autoimmune disorders are diseases caused by immune responses against autologous tissues, and autosomal dysregulation regulated by granulysin refers to <autoimmune disorders associated with abnormally high or high activity of the granulysin gene. For example, these disorders include Steven Johnson syndrome, toxic epidermal necrosis, transplant versus host disease, Beth's disease, ankylosing spondylitis, systemic lupus erythematosus, dermatomyositis, polymyositis And organ transplant rejection. ,

I'm using a biological sample taken from a test individual to contact a detectable protein or a nucleic acid encoding a granulocyte (eg, VIII and genomic 01^8) to assess particle dissolution in the biological sample. The presence or absence of a protein or nucleic acid or the amount of expression, so that the protein or nucleic acid of the granulysin present in the biological sample can be detected. The term "biological sample" includes tissues and cells isolated from the individual.肇 and biological fluids, as well as tissues, cells, and biological fluids in the body, #乂佳的 biological samples are serum or vesicles. There are many ways to measure the amount of granulysin gene, including but not limited to: measurement by particle dissolution mRNA encoded by the gene; measuring the amount of protein encoded by the granulysin gene; or measuring the activity of the protein encoded by the granulysin gene. The mRNA expression of the granulysin gene in the cell can be in the form of an in situ or in vitro experiment. Determination, for example, by -cell extraction 13 willow 808973 ~ Γ ^ ΝΑ can be used for hybridization or amplification analysis, including but not limited ^Southern or Northern (N〇rthem) dot method, _e arrays. The method of side mRNl 2 preferably includes separating the mRNA from a nucleic acid molecule (ie When the probe is in contact, the nucleic acid molecule can be hybridized with the base (4), mRNA, to be detected. For example, the nucleic acid probe can be a full-length granulated lysin nucleic acid or a granule lysin-age protein. Such as a length of at least 7, 15, 30, 50, 100, 250, or 500 nucleotides (nude〇tides) and sufficient specificity to meet the granulysin mRNA or genomic QNA under severe conditions ( Genomie DNA) hybridizes to an oligonucleotide (〇lig〇nUcleotide) and the probe can be planted at a fixed matrix address. Other probes suitable for use in assays are described herein. One type is to immobilize mRNA (or cDNA) on the surface and then contact the probe, for example, to run off her shame, and then transfer the mRNA from the colloid to a transfer membrane, such as nitrocellulose. Another type is to fix the probe to the surface.

The mRNA (or cDNA) is then contacted with the probe, such as the two-dimensional gene wafer microarray described below. A person skilled in the art may suitably use a conventional mRNA assay to detect the mRNA expression of a granulysin gene. 14 200808973 The amount of mRNA expression encoded by the granulysin gene in the sample can be assessed using nucleic acid amplification, such as the instant polymerase chain reaction (Mullis (1987) US Patent No. 4,683,202), ligase chain reaction (Barany) (1991) Proc. Natl Acad Sc/· C/&4 88:189-193), seif sustained sequence replication (Guatelli et al, (1990) Proc. Natl Acad· Sci·87:1874 -1878), transcriptional amplification system (Kwoh et al(1989), Proc. Natl Acad. Sci. USA 86:1173-1177) - Q-Beta Replicase (Q-Beta Replicase) Lizardi θ a/·, (1988) stealing/7^%>/qing 6:1197), rolling drcle replication (Lizardi as α/·, US· Patent No 5,854,033) or any other The nucleic acid amplification method is followed by detection of the amplified molecule using conventional techniques. An amplification primer used in the present invention is defined as a pair of nucleic acid molecules (either positive and negative, respectively, and vice versa) that can be annealed to the 5' or 3' region of a gene, and both There is a short piece between them. Typically, the amplification primers are about 10 to 30 nucleotides in length and about 50 to 200 nucleotides in length on the left and right sides. The primer can amplify a nucleic acid molecule comprising a nucleotide sequence flanking the primer under appropriate conditions and an appropriate reagent. An in situ method is to prepare/process a cell or tissue sample, and then fix the sample on a support, such as a glass slide, followed by an mRNA that encodes the lysin gene to be analyzed. The combined probes are subjected to a heterozygous reaction. 15 200808973 In another embodiment, the method further comprises contacting a compound or reagent that detects granulysin mRNA or genomic DNA with a control sampie, and comparing the presence of the control sample with the subtracted sample The amount of granulysin mRNA or genome expression. In yet another embodiment, described in U.S. Patent No. 5,695,937 = Serial Analysis of Gene Expression for Transcription of Side Granulysin

There are many ways to determine the amount of granulysin protein expression. These methods include contact with reagents that bind to granulysin proteins or their antigens or immunogenic gene fragments, such as and Touch to assess the amount of protein in the sample. Further, in the examples, the antibody of the sample has - can be detected. The antibody can be a plurality of antibodies, or preferably a monoclonal antibody.

Segments are available, or a)2. “—The term means that the probe or antibody comprises a substance that binds to the probe or antibody (eg, physically binds), and j ===_; simply reacts with the cleavable substance to indirectly "remember the probe or Antibody

An example of a substance. M, W,. The detailed interview method can be _ in vitro recording and internal inspection (milk vm?) detection of biological _ in vitro experiments with (4) (10) in the poor lysin protein, the basket is used for the detection of granules Baibei's technology includes enzyme-linked immunosorbent assays for free adsorption knife analysis (e Qingmei 200808973)

ELISAs), immunoprecipitations, immunofluorescence, enzyme immunoassay (EIA), radioimmunoassay (RIA), and Western blotting (Westem w〇t analysis) ). In vivo experiments for detecting granulysin proteins include introducing a labeled anti-granulolytic antibody into a body, for example, the antibody can be labeled with a radioactive marker, the marker of which can be Standard imaging techniques detect its presence or location in an individual. In the case of a sample, the sample is labeled, such as biotinylated, followed by contact with an antibody, such as an antibody on an antibody array. The granulysin antibody can be detected by a avidin such as a binding-light-labeling. In another embodiment, the method for detecting a granulin protein further comprises contacting a sample of a dragon with a compound or reagent capable of detecting a granulin protein, and comparing the particles dissolved in the sample of the dragon and the sample. The amount of protein. The invention also includes a kit for detecting the presence of a granulysin in a biological sample, for example, the test kit comprises a compound of a peptin protein or A or a pre-loaded orbital or touch Subtraction to a suitable group of detection _ heart 2 test test mosquitoes - steps include the use of the set, , ... ... v-protein or nucleic acid instructions. 17 200808973 An antibody-based test kit comprising: · (丨) ____ sub-antibodies, such as 依 _ _ hiding, the 尬 可 can be combined with a multi-peptide corresponding to the marker of the present invention, and Optionally (2) a second and different antibody 'the secondary antibody is not bound to the multi-peptide or to the primary antibody' and the secondary antibody is linked to an agent detectable. The test reagent set based on the voucher core includes: (1) a nutrient nucleotide, such as a detection-labeled oligonucleotide, which can be hybridized with the -nucleic acid, The nucleic acid phase encodes a multi-peptide, which is equivalent to the label of the present invention, and the S(2) pair is introduced, and a nucleic acid molecule corresponding to the label of the present invention is amplified. The test kit may also include a buffer reagent, an acid reservoir, or a protein f stabilizing reagent, and the test reagent kit may also include a necessary component (such as an enzyme or a receptor) for detecting the detectable reagent. quality). The test kit can also include one or a series of samples that can be compared for comparison with the sample handle. Each test of the test _ 纟 can be sealed in a single load, and all the different cranes are placed in a single-package, and the test results of the test kit analysis results. The test methods described herein can confirm that an individual has, or has a poor, abnormal, or unnecessary manifestation or activity-related =: or disorder. Here, "unwanted" means containing unnecessary phenomena in biological reaction tissues, such as dysregulation of cytotoxic tau cells (such as Steven's Johnson & Johnson syndrome, toxic epidermal necrolysis or transplantation against host disease). ) 18 200808973

In the examples, it was confirmed that the granulysin was abnormal or unnecessary, or the activity was abnormal. From - individual acquisition - test samples, and then evaluate the protein or nucleic acid of granulysin (such as mRN genomic DNA), in which the amount of protein or nucleic acid of granulysin is 1 'testable - individual suffering from money _ granulysin Abnormal, or unnecessary, _ active phase _ risk of disease or disorder. Here, the "test sample" - the word means - the biological sample of the body - containing - biological fluid (such as blister or serum), cell samples, 'the analytical method described here, can be touched - Was given -^^ (,_^#J(ang〇nist) .#^#J (a,tag〇nist) ^ pseudo-peptide (p_Gmi reward), protein f, occupation, nucleic acid, small molecule, Or other drug candidates) or accepting - cells, - tissue or a treatment of a disease - whether an adverse drug reaction or other loss of the drug, such as transplantation against host disease.

The other characteristic of this indication is that the square of the sample taken from one body is calculated. =: The method includes a gene expression map providing one of the gamma samples. The 丝/, the middle silk spectrum contains the present value of the amount of the silk. Further: The step contains a value of 1 曰, such as a composite value, compared with the reference value or the base value. The gene expression profile in the aforementioned samples can be obtained by any method (e.g., provided by the sample - nucleic acid, and then the array is contacted). This method can be used to detect dysregulation of sputum cell regulation in a body, wherein an increase in the amount of lysin 19 indicates that the individual has or is predisposed to the disorder. In addition, the method can be used to monitor the treatment of a disorder with a body disorder, for example, a gene expression map is taken from a sample of a ship after the treatment, and the gene table = map can be correlated with - baseline or _ from pre-treatment or disorder Symptoms of other individuals with symptoms (see example G〇lub d 286:531) 〇· « Μ &amp; ΙΙϋ / As discussed, granulysin is involved in the regulation of toxic killing of tau cells, in this case, granulysin inhibitors Can be used to treat this type of disorder. The present invention provides a method for identifying a dysregulated granules of a glutathione inhibitor for the treatment of cell cytotoxicity, which may be from a commercial supplier or according to the methods described below or any of the conventional methods. Candidate compounds (such as proteins, peptides, peptoids, peptoids, antibodies, small molecules, or other drugs) can be obtained by a variety of methods using a known integrated database, including: Peptide database, test derivative (4) library (molecular database with function of function 'but a new, non-Sheng Yuetai framework, can resist the degradation of enzymes), workable parallel solid phase or liquid phase data Library, synthetic database screened by deconvolution or affinity chromatography, and a particle-one compound database (〇ne seven ead 〇ne_c〇mp〇und libraries) 〇Zuckermann et al 1994, J. Med Chem 37.2678 2685, and Lam, 1997? Examples of synthetic methods for the Anticancer Drug Des. HI45 ” sub-database can be found in 200808973 1993, PNAS USA 90:6909; Erb et aL, 1994, PNAS USA 91:11422; Zuckermann «/., 1994, J. Med. Chem. 37:2678; Cho et al., 1993, Science 261:1303; Carrell et al., 19945 Angew. Chem. Int. Ed· Engl. 33:2059; Carell et aL, 19945 Angew Chem. Int. Ed. Engl. 33:2061; and Gallop et al., 1994 J· Med· Chem·37·1233. A library of compounds can be presented in solution (see Houghten, 1992, Biotechniques 13:412_42ί), on magnetic beads (see Lam, 1991, Nature 354: 82-84), wafers (see Fodor, 1993, Nature 364: 555-556), bacteria (see U.S. Patent No. 5,223,409), spores (see U.S. Patent No. 5,223,409). plastids (see Cull β β/·, \ΐ992, PNAS USA 89:1865-1869) Or bacillus (see Scott and Smith 1990, Science 249: 386-390; Devlin, 1990, Science 249: 404-406; Cwirla et al, \99^ PNAS USA 87: 6378-6382; Felici 1991, J. Mol · Biol. 222:301 _310; and US Patent No. 5,223,409) To confirm the granulysin inhibitor, a candidate compound can be contacted with a system containing granulysin. The system can be a cell-free system or a cell system (cell_c〇ntaining system), such as a cell line model for in vitro experiments or an animal model for in vivo experiments. In a cellular system, the cell may naturally exhibit a granulysin gene or may be modified to express a recombinant nucleic acid comprising a particle > glutathione gene coding region fused to a heterologous promoter 'or granule lysate The gene promoter sequence fuses a receptor (recept〇r) gene. The amount of granulysin gene expression can then be measured. The amount of granule lysin gene expression can be determined by measuring the amount of mRNA or protein, and the amount of mRNA expression in a tissue sample or body fluid is measured using conventional techniques. In order to measure the amount of mRNA, the cells can be firstly dissolved, and the amount of _ Α in the RNA obtained by purifying or semi-chemically lysing the lysate or the lysate from the cell lysate can be measured by the known technique. Analytical method (using gene-specific DNA or RNA without a marker that can be detected) and quantitative or semi-quantitative RT_pCR (using appropriate gene-specific primers). Alternatively, quantitative or weight in situ analysis can be performed using tissue sections or suspensions of undissolved cells and DNA or probes that are detectable (e.g., fluorescent or enzymatic). More mRNA stereophony methods include the RNase protection assay (RPA) and the Serial Analysis of Gene Expression (SAGE). Methods for measuring the amount of protein in tissue samples or body fluids are also known techniques, and many such methods use antibodies (e.g., single or multiple antibodies) to specifically bind to the target protein. In such assays, the antibody itself or a secondary antibody to which it binds has a detectable label. In addition, the antibody binds to biotin and uses a detectably labeled avidin (a multi-peptide that binds to biotin) to detect the presence of biotinylated antibodies. Combining these methods (including multRayer sandwich assays) can be used to improve the sensitivity of the overall method. Some of the aforementioned protein assays (such as enzyme-linked immunosorbent assay or Western blot) can be used to determine body fluids or cell lysis. Other protein assays (such as immunohistochemical methods or fluorescence flow cytometry) can be used for tissue sections or suspensions of undissolved cells. The method of measuring the total amount of labeling utilizes the nature of the label and the known techniques. Suitable labels include radioactive materials (eg, 125, %, 35S, 3 or 32p), and enzymes (such as alkaline phosphatase (he also phosphatase). ), horseradish peroxidase (horsefrash i • peroxidase), lumifolium (ludfefase) or galactosidase (cold-glactosidase), fluorescent substances or proteins (such as luciferin _ ( ^uorescein ), if Danming (1^1〇(1&amp;111丨1^), Phycoerythrin, green flu〇rescent protein (GFP), fluorescent protein (blue f)Uprescent pr 〇tein, BFP)) or illuminant (as provided by Quantum D〇t Corporation, Palo Alto, CA (^(^ nanoparticle). Other applicable analytical methods include 疋1 immunosuppressive hall (immUn〇preCipi plus I〇n) or complement fixation assay. 10 To determine the ability of a candidate compound to inhibit granulysin, the method described by W can be compared with the control group without the candidate compound. Measured amount or activity is low In the control group, it can be determined that the candidate compound is effective for treating dysregulation of cytotoxic cytokine regulation. Further, the animal model can be used to confirm the efficacy of the previously determined effective substance, for example, to confirm that a compound can be used for treatment. The cytochrome T cell sculpt can be administered to the granule/nuptamine squirrel (nud miee), and the following example is given. 23 200808973 PRODUCTION == conjugated to granulysin The method of the antibody is not specifically limited: and the source of the multi-drug antibody and the monoclonal anti-antiserum is a protein having the present invention - an immunized animal, such as a rabbit, and all kinds of multi-strain and monoclonal antibodies, human antibodies And humanized antibodies made by genetic engineering are also included. The above "antibody," - means - immunoglobulin molecule or its immunological activity 14. Sickle, ie an antigen-binding part, refers to A protein contains at least one, preferably two heavy (variable regions) V< VH) and at least one, preferably two light (variable regions) VL), the heavy chain variable region and the light chain variable region can be further subdivided into highly variable regions, that is, complementary region determining regions (CDRs), and preferentially dispersed regions, that is, constructing The length of the region (framew〇rk regions ' FR) tectonic region and the complementarity determining region has been precisely defined in the past (see Kabat β β/· (1991) 〇/

Proteins of Immunological Interest, Fifth Edition

Department of Health and Human Services, NIH

Publication No. 91-3242, and Chothia ei fl/. (1987) J. Mol·Biol. 196:901-917). Each heavy chain variable region and light chain variable region is composed of three complementary determinant regions and four structural regions, ranging from 24 200808973 version of the amino-terminus to the carbyl Xy_terminus. The sequence is as follows: tectonic region 1, complementarity determining region, tectonic region 2, complementary crest region 2, tectonic region 3, complementarity determining region 3, and tectonic region 4〇μ. The antibody may further comprise a heavy chain and a light chain invariant region. ^ (constantregion), thus forming an immunoglobulin self-heavy chain and an immunoglobulin light chain, respectively. The heavy chain invariant region contains three regions, CH1, CH2, and CH3, and the light chain constant region contains only one region of C£. The aforementioned heavy and light scale variable regions comprise a binding domain that binds to the antigen. The invariant regions of the aforementioned antibodies substantially modulate the binding of the anti-sense to host tissues or factors, including cells of various immune systems (e.g., effector cells) and the first of the standard complementary systems. Component (Clq). The term "immunoglobulin" as used herein refers to a protein consisting of a multi-peptide encoded by one or more immunoglobulin genes. The officially recognized human plague globulin gene includes κ, a, I (IgAl and IgA2), 7 (IgGl, IgG2, IgG3, IgG4), 5ε and from a constant region gene, and myriad immunoglobulin variable region gene. The full length immunoglobulin light chain (about 25KDa or 214 amino acids) is It is encoded by the carboxy terminus of the variable region gene amino terminus (about 110 amino acids) and the 1 or λ invariant region gene; the full length immunoglobulin heavy chain (about 5 〇 KDa or 446 amino acids) is also The antigen-binding fragment of the antibody is encoded by a variable region gene (about 116 amino groups 25 200808973 S〇 and one of the other invariant region genes, such as Τ (encoding about amino acid). Or, the term "antibody portion", or, "I") means that one or more fragments of a full-length antibody retain the ability to specifically bind to the antigen, such as EGFR or CDS polypeptide or a fragment thereof. For example, the antigen-binding fragments of the 'body' include, but are not limited to: 1) A Fab fragment, a monovalent fragment consisting of a heavy chain variable region, a light chain variable region, a light chain invariant region, and a heavy chain invariant region; (2) F(ab)2 fragment The 'system τ contains a bivalent fragment of two Fab fragments linked by a double-disc bridge (the center of the bridge); (3) a fragment of a ke, consisting of a heavy chain variable region and a Cm region of a heavy chain constant region ; ' (4) &amp; fragment, consists of a light-key and a heavy-bond variable region of an antibody 単 arm, · (5) - dAb fragment (see Ward as β /, (1989) 341:544-546 ), consisting of a heavy chain variable region region; and (6) — an isolated complementarity determining region. Furthermore, although the heavy and light chain variable regions of the ^^ fragment are encoded by different genes, In the &amp; fragment, the two can be recombined to form a single protein chain by a synthetic linker, wherein the heavy and light chain regions are paired to form a monovalent molecule (known single-chain Fv (scFv); see Examples of Birds are 〆 (1988) 242: 423-426; and Huston et al (1988) Proc. Natl (10) 85: 5879-5883). Such single-chain antibodies are also included with the antigen of an antibody. These antibody fragments are familiar to the skilled artisan (10), Zhizhilou, and the same method of screening for the same way as the intact antibody. 26 200808973 A suitable antibody can be a monoclonal antibody, In one embodiment, the antibody may be a recombinant product, such as a product obtained by phage expression or by a combination method. The use of phage expression and combination methods to produce antibodies is a conventional technique (see Ladner US Patent No. 5,223,409; Kang ei aL International Publication No. WO 92/18619; Dower et al IntemationalTublication No, WO 91/17271; Winter έί a/. International Publication WO 92/20791; Markland et al. International Publication No. WO 92/15679; Breitling et Al International Publication WO 93/01288; McCafferty et al. International Publication No. WO 92/01047; Garrard et al International Publication No. WO 92/09690; Ladner et al. International Publication No. WO 90/02809; Fuchs et al ( 1991) Bio/Technology 9: 1370-1372; Hay et al. (1992) Hum Antibod Hybridomas 3:81-85; Huse et al (1989) Scienc e 246: 1275-1281; Griffths et al. (1993) EMBO J 12: 725-734; Hawkins et al. (1992) JMol Biol 226: 889-896; Clackson et al (1991) Nature 352: 624-628; Gram et al. (1992) 89:3576-3580; Garrad ei al (1991) Bio/Technology 9:1313-1311 \ Hoogenboom et aL (1991) Nuc Acid Res 19:4133-4137; and Barbas et al. (1991 ) Corpse 88: 7978-7982, the entire contents of which are incorporated herein by reference. In one embodiment, the anti-system is a complete human antibody (such as an antibody produced by a mouse, which is produced by genetic engineering using human immunoglobulin sequence 27 200808973), or a non-human antibody. Such as a rodent (mouse or rat), sheep, primate (such as monkey), Lujun antibody, and preferably a non-human anti-systematic antibody (mice or rat antibody) . Methods for the production of rodent antibodies are known in the art. « Human monoclonal antibodies are produced in a transgenic mouse with a human immunoglobulin gene, which is superior to that produced by the mouse system. The spleen cells of a gene-transferred mouse that produces an immune response with a specific antigen produce a fusion tumor. Tumors secrete a single antibody with a specific affinity for a human protein anti-swallowing epitope (see Wood β a/· International Application WO 91/00906, Kucherlapati et al PCT publication WO 91/10741; Lonberg et al. International Application WO 92/03918; Kay et al International Application 92/03917; Lonberg, N. et al. 1994 368: 856-859; Green, LL et al. 1994 Nature Genet. 7:13-21; Morrison et al 1994 Proc. Natl Acad, ScL USA 81:6851-6855; Bruggeman et al. 1993 Year Immunol 7:33-40; Tuaillon et al. 1993 PNAS 90:3720-3724; Bruggeman et al. 1991 Eur J Immunol 21:1323-1326) The variable region or part of the antibody, such as the complementary variable region, can be produced by a non-human organism, such as a rat or a mouse. Among them, chimeric, that is, complementary variable region transplantation, and humanized antibodies can be utilized. Non-human organisms, such as rats or mice, can reduce the antigenicity in humans during the present month, as in the case of variable frame or invariant regions, resulting in anti-limb modification. Grafted antibodies can be produced by conventional recombinant DNA techniques, for example, 'a gene encoding one of the murine (or other species) monoclonal antibody molecules encoding an Fc invariant region is first removed by a restriction enzyme, a murmur-like Fc region, and then Substituted by a corresponding gene encoding a human Fc invariant region (see R〇bins〇n et α/.5 International Patent Publication PCT/US86/02269; Akira, et al, European Patent Application 184, 187; Tamgi^hi, M· European Patent Application 171,496; Momson et al, European Patent Application 173,494; Neuberger et&quot;al, International Application WO 86/0Γ533; Cabilly et al US Patent No. 4?8169567; Cabilly et al, European Patent Application 125,023; Better et at (1988 Science 240: 1041.1043); Uu et al. (1987) PNAS 84: 3439-3443; Liu et al, 1987, 1 Immunol 139: 3521-3526; Sun e/a/. (1987) PNAS 84:214 -218; Nishimura et al., 1987, Cane. Res. 47:999-1005; Wood et al. et al (1985) Nature 314:446-449; and Shaw β α/·, 1988, J. C(10)cer /add 80:1553-1559) Antibody with humanized or complementary region There are at least one or two, but usually three recipient complementarity determining regions (heavy and/or light immunoglobulin chains) are replaced by a supplier complementarity determining region, the antibody being at least partially non-human complementarity determining regions Substituted or only a few complementary 29 200808973 Ant colonies will be replaced by non-human cross-relationships, and only t replaces some of the complementarity determining regions necessary for binding to human antibodies or human scorpions, ^ Cap better supplier of murine antibodies For example, a rat or mouse antibody, and a preferred recipient is a human entanglement or a human consensus framework (_ensus framework). In general, an immunoglobulin that provides a complementarity determining region is said to be a donor, and an immunoglobulin/white that provides a framework is referred to as a recipient. In a consistent application, the donor immunoglobulin is non-human (such as a rat), and the frame is a natural frame (such as a human) or a common frame, frame, or a similarity of at least about 853⁄4 or higher. Preferably, the sequence is 9〇%, 95%, or higher. In the present invention, the so-called consensus sequence refers to the amino acid (or nucleotide) having the highest frequency of occurrence in the family of related sequences. (See Wi ker5 Fr face Genes to Clones (Verlagsgesell, Weinheim, Germany 1987)). In a family of proteins, all positions in the consensus sequence are occupied by the most common amino acids in this family in the family. The two amino acids appear at the same frequency, and both are included in the consensus sequence. A consensus framework means the framework regions in the same immunoglobulin sequence &gt; column. An antibody can be humanized by conventional techniques, and the production of a humanized antibody can be carried out by substituting an Fv variable region sequence which is not directly contained in an antigen that binds to an equivalent sequence derived from a human Fv variable region, and is generally produced. For methods of humanized antibodies, see Morrison, S. L" 1985, 229.1202-12075 by 〇i et aL, 1986 ^ BioTechniques 4:214,

And by &lt;RTIgt;&quot;&quot;&quot;&quot;&quot;&quot;&quot;&quot;&quot;&quot;&quot;&quot;&quot;&quot;&quot;&quot; The method comprises isolating, processing, and expressing a nuclear sequence encoding all or part of an immunoglobulin Fv variable region from at least one of a heavy chain or a light chain, the source of the nucleic acid being a technique known to those skilled in the art. For example, the nucleic acid can be obtained from a fusion tumor capable of producing an antibody to a multi-peptide or a fragment thereof of interest. The humanized antibody, or a fragment thereof, encoded by the recombinant DNA can then be cloned into an appropriate expression vector. &lt;Humanized antibodies can also be fused to a framework in which the amino acid (10) is added. Amino acids are substituted in the framework region, which enhances the junction with the antigen'. For example, a humanized antibody has a frame residue with the donor or another frame other than the recipient. A framework amino acid residue identical to the amino acid other than the amino acid residue. In order to generate such antibodies, a selection of humanized immunoglobulin chains and a small number of recipients of the amino acid can be replaced by the corresponding donor silk. Preferred substitution positions include amino acid residues immediately adjacent to the complementarity determining region, or may be complementary to (4) the other side of the acid, and the standard amino acid of the amino acid selected from the supplier, U.S. Other techniques for humanized antibodies are described in Padlan over α/· EP 5丨9596 A1. Methods of Treatment The present invention also provides methods of treating one or more of the above disorders, from which an individual to be treated can be identified by the general detection technique of such disorders. The individual may optionally determine the expression or activity of the granulysin polypeptide gene, and if the gene expression or activity in the _ body sample is higher than the substrate expression or activity of the 200808973^ sample towel, then the system A candidate for treatment with an effective dose of a granulysin inhibitor. The term "treating" as used in the present invention means administering to a subject having one or more of the above disorders a compound for treating, alleviating, alleviating, preventing or improving the symptoms, the symptoms of the disorder, the number of times of the disorder. The disease is sad, or the tendency to occur. By analogy, the effective dosage, the term oxime and the amount of the compound produced, in the individual treated as described above, can produce medically satisfactory results. Treatment can be performed in vivo (Wvo) 1 or ex vivo (ex vzvo), with or without other drugs or treatments. f In the in vivo treatment method, a granulysin inhibitor is administered to a body. Generally, the compound is uniformly mixed with a pharmaceutically acceptable carrier (such as physiological saline), and then administered orally, Intravenous infusion, or subcutaneous, intramuscular, intramedullary, intraperitoneal, intrarectal, intravaginal, intranasal, intratracheal or intrapulmonary injection or implantation, for the treatment of skin disorders, Such as Steven's Johnson & Johnson syndrome and toxic epidermal necrolysis, the compound can be administered topically. The dosage required will be determined by the route of administration chosen, the nature of the formulation, the type of disease, the size, weight, surface area, age, and sex of the individual, the administration of other drugs, and the judgment of the attending physician. The preferred dose is in the range of 〜·〇1~l〇〇mg/kg. The difference in the required dose is estimated by observing the type of effective compound and the efficacy of different routes of administration. For example, oral administration compared to intravenous Injections are expected to be administered at a higher rate of 200,808,973 and the difference in these agents 1 can be judged by generally accepted empirical practices. Encapsulation of the compound in a suitable vehicle (e.g., polymeric micromolecules or implanted devices) can increase the efficacy of administration, particularly oral administration. In addition, a plurality of nucleotides comprising a nucleic acid sequence encoding a granulysin inhibitor can be administered to a body which encodes an anti-particle lysin antibody, an antisense RNA (anti-ser^e RNA), or a small fragment of interfering RNA (such as an RNAi test), targets granulysin and inhibits the appearance or activity of granulysin. ♦&gt;"RNAi" or "RNA interferon" means that a sequence-specific or selective process results in down-regulation of a target molecule (such as a target gene, protein or RNA) within the scope of the present invention. The degradation characteristics of RNA molecules (such as in cells) using RNAi, and their degradation is catalyzed by RNA-induced silencing complex (RISC) enzymes. RNAi occurs in the cells and naturally removes foreign substances. RNAs (eg virus). Natural RNAi begins with a fragment that is cleaved by a direct degradation mechanism from a double-stranded rna. In addition, RNAi can also be artificially initiated, for example, the expression of a silent target gene. By "RNAi agent" is meant that an RNA (or an analog thereof) has sufficient sequence to complement a target RNA (e.g., degraded rna) to directly perform RNAi. An RNA reagent having sufficient sequence to complement the target RNA to directly perform RNAi means that the (iv) eight reagent has a sequence sufficient to cause destruction of the target RNA by an RNAi device (e.g., an RNA-induced silencing complex) or a process. An rna reagent has sufficient sequence to complement the target RNA to directly perform RNAi, and the RNA reagent has a sequence sufficient for inhibition of translation of the target RNA by a gastric fistula device or process, and an rna reagent can also have a sequence. A sequence sufficient to complement a target rnA encoded by the target DNA sequence to silence the chromosome of the target DNA sequence. In other words, the RNA reagent has a sequence sufficient to cause inhibition of the transcriptional gene, such as down-regulating gene expression at or near the target DNA sequence, and causing chromatin (chf〇mkin) at or near the target DNA sequence. Structural changes. A ribonucleic acid molecule means a polymer of a ribonucleotide, and a DNA or DNA molecule or a deoxyribonucleic acid molecule means a deoxygen nucleus. A polymer of glycosides. DNA and RNA can be naturally synthesized (e.g., by DNA replication or DNA transcription, respectively), and rna is post-transcriptionally modified. DNA and RNA can also be

By chemical synthesis, DNA and RNA can be single strands (such as ssDNA and ssRNA, respectively) or multiple strands (such as double strands, ie dsDNA and dsRNA, respectively). Administration of the polynucleotide can be achieved by using a conventional polymer, a biodegradable microparticle or a microcapsule administration device. Another method of achieving nucleic acid uptake is the use of microlipids prepared by conventional methods which can be incorporated separately into these 200808973 vehicles or in conjunction with tissue-specific antibodies. In addition, a molecular chelating agent composed of a plastid (fine nid) or a simplification thereof (her 〇) can be prepared, and the molecular age _, by electrostatic or covalent force, and poly-L- Lysme) ligated, poly- lysine can be combined with - can bind to target cells (see Cristi, λ, 1995, τ. Mol. Med. 73: 479). Correction, can use the expertise of the organization The transcriptional regulatory component targets it to a specific tissue. Transferring naked _8 (v without carrying jade) to the muscle, subdermal or subcutaneous is another way to achieve in vivo performance. A polynucleotide, such as a performance vector, a nucleic acid sequence encoding a lysin inhibitor is operably linked to a promoter or an enhancer-promoter conjugate. Suitable expression vectors include plastid and disease vectors, such as Herpes vimses, retroviruses, vacciniaviruses, attenuated vaccinia viruses, canary ph〇χ vimses, line viruses (5) Qiu (10) Such as milk) and line related diseases (adeno_ Associated viruses ). It is well known to those of ordinary skill in the art subject area that the need for a patient to be informed is determined by a number of factors in the uranium. Although the dosage administered is varied, the preferred dosage for administration of the polynucleotide is from 1 to 6 to 12 copies of the polynucleotide molecule, which may be administered if necessary. And the route of administration can be any of the routes listed above. 35 200808973 In ex vivo (ex vfvo) treatment, treatment of individuals with dysregulated tau cell regulation disorders, including transfection or transduction of polynucleotides encoding granulysin inhibitors a cell obtained from the individual, which is transfected into a vector via an in vitro experiment (9) v/iro) designed to be upstream of the transcription initiation site of the endogenous granulysin inhibitor gene naturally produced in the cell genome Inserting a new and active promoter in a homologous recombination process. After selecting and amplifying a cell exhibiting a granulysin inhibitor to a desired value, the transfected or transduced cell is returned to the individual. in. For example, the cells may include nerve cells, hematopoietic cells (such as bone marrow cells, macrophages, monocytes, dendritic cells, T cells, or B cells), fibroblasts, epithelial cells, endothelial cells. A keratinocyte, or a muscle cell, as long as the cell can survive in the individual, the cell can be used as a source of a granulysin inhibitor. The method of ex vivo treatment (ex Wvo) includes the steps of obtaining cells from one body, culturing the cells, transducing the cells with the expression vector, and maintaining the growth of the cells in an environment suitable for exhibiting a granulysin inhibitor. The transduction step is accomplished by any general method using in vitro gene therapy, including calcium phosphate, lipofection, electroporation (eiectroporati〇n), and viral infection (Cviral infection). And biolistic gene transfer. In addition, vesicles or polymeric micromolecules can also be used, and cells that have been successfully transduced are screened again, for example, screening for granules 36 200808973 == inhibition _ cells, followed by nucleus or transplantation A right-invented invention also includes a packaged product, including a carrier, a granulysin inhibitor, and a carrier-containing product, which is a gas to the inhibitor to treat a T cell. The pharmaceutically acceptable carrier is mixed with the pharmaceutically acceptable carrier, the medicinal agent, the stimulating agent, the dispersing agent, the antibacterial and antifungal agent, the osmotic and delayed absorption agent. The system can be tailored to the desired level, for example, as a capsule, a gel, or a second dose (four). The lysine may contain any freshly-received, acceptable substances such as gelatin or cellulose; the configuration may be in accordance with conventional methods, ie, the inhibitors and solid carriers and - Lubricating surface blending, and the solid carrier may include temple powder and sugar bent〇nite. In addition, the administration form of the inhibitor may be - a hard shell or a binder, a storm (such as lactose or mannitol (mannitGl)), a commonly used filler, and - interview #j. And the side of the object can be administered via a parenteral route, and examples of parenteral dosage forms include aqueous solutions with active agents, isotonic physiological saline or 5% dextrose, or other known pharmaceutically acceptable excipients. . Cyclodextrin (4) - Coffee (8) or 37 200808973 Other soluble reagents are generally available to those skilled in the art for the administration of therapeutic agents. The composition of the composition is a pharmaceutical composition comprising a pharmaceutically acceptable carrier and an effective dose of a lysin inhibitor, and the city can be used to treat poisonous killing 7 cells. Controlling the imbalance. The pharmaceutically acceptable carrier includes a solvent, a aliquot, a film coat, a anti-set agent, and an osmotic and delayed absorption agent. The present invention can be formulated into a desired dosage form, for example, a capsule/spinner, or an oral lozenge. Wherein the capsule may comprise any standard pharmaceutically acceptable substance, such as vegetable collagen (gelatln) or animal collagen (cdl_ = method according to the towel, that is, the combination of the secret and the surface carrier and a Run Yue Tong, W Examples of the carrier of the diaphores include the powder of the temple and the sugar viscous, and the type of the stagnation lj can also be -1, the role of the preparation (such as recording or nectar gamma-fine, and - ingot type reagents. Examples of inhibitors that can be administered via non-intestinal 'non-gastrointestinals include physiological saline with equal activity = equal permeability or 5% glucose, or other known, orally acceptable excipients, cyclodextrin or other solubles. Reagents 具有 those of ordinary skill in the art are known to be useful as excipients for the administration of therapeutic agents. 38 200808973 The composition of the present invention also includes a safe and effective dose suitable for skin area. The pharmaceutically acceptable carrier can be administered to the skin at a suitable concentration, and the carrier can act as a diluent, a dispersing agent, a solvent or a pharmaceutically acceptable carrier. Active substance The mass may be at a suitable concentration and the reagent distributed at the point of consumption, and the type of the carrier may be a solid, semi-solid, or liquid '4 good looking _ type Wei Yu, New, 4 gel, More preferably, it has the characteristics of sufficient concentration or can avoid the active substance sinking. The dialysis itself can be secretive or has a beneficial effect on the skin. The shellfish can be completely chemically and in a chemical form. The essential components are phased and not excessively detrimental to the stability of the composition, efficacy f, and other related benefits.

... This is a fortune-making. The composition used in the individual can be made into a widely known and multi-formed form of the product including, but not limited to, emulsion, gelatinous, gelatinous, mucus-like, ointment. Forms, pastes, and vesicles/tops, which are excavated by Xuxiang lions, including but not limited to solution, aerosol, emulsified, gel 2 solid, and vesicles. / ▲ ▲ preferred carrier may comprise a pharmaceutically acceptable hydrophilic dilution: a combination of hydrophilic diluents including water, such as Crc4 monohydric alcohol and low molecular weight ethylene glycol and polyol (including propylene glycol, Polyethylene glycol 'such as molecular weight 200 to 600) organic hydrophilic diluent, poly 39 200808973

An oil emulsion in water or an aqueous emulsion of oil, such as a water-in-silicone emulsion, typically containing from about 1% to about 50% of oil emulsion (preferably from about 1% to about 1%) 30%) of the dispersed hydrophobic phase and from about 1% to about 99% (preferably from about 40% to about 90%) of the continuous hydrophilic phase; and typical aqueous water emulsions contain from about 1% to about 98% (preferably From about 40% to about 90%) of the dispersed hydrophilic phase and from about 1% to about 5% (preferably from about 1% to about 30%) of the continuous hydrophobic phase. In addition, the emulsion is also included = alcohol to 2.25), glycerol, butanediol, mountain-i-axis, 18-isopropanol, xylose, ethoxylated, and its compounds The forgery is preferably a hydrophilic diluent containing at least (iv). &quot;The limbs, and have a = Bu., the recorded carrier also contains - milk simple, and the milk $ human, - ^ 'especially 疋 - aqueous phase, and - hydrophobic phase, such as lipid, oil, or 3 oil Property. As is generally known in the field of tobacco, the hydrophilic components of the hydrophobic phase are dispersed in the hydrophobic lion, or vice versa, forming a dispersed phase or a continuous phase of hydrophilic or hydrophobic water, respectively. By disperse phase is meant that small molecules or droplets are dispersed in a continuous phase towel and surrounded by a continuous phase which is also known as an internal phase or a discontinuous phase. The emulsifier may be or comprise (eg, in a three-phase or other multi-phase emulsion) a gel network, as described in GM Eccleston Application of Emulsion Stability Theories to Mobile and Semisolid 0/W Emulsions, Cosmetics &amp; Toiletries, Vol. 101, November 1996, pp. 73-92, incorporated herein by reference. Preferred compositions in the present invention are oil-in-water emulsions. A preferred embodiment of the topical composition of the present invention has an apparent viscosity r (-arent viscosity) of from about 5,000 to 200,000 mPa.s (centipoise), for example, a preferred emulsion form has The apparent viscosity is from about 10,000 to 40,000 mPa.s; and the preferred paste form has an apparent viscosity of from about 30,000 to 160,000 mPa.s. Viscosity • is determined using a viscometer (Brookfield DVII RV), TD spin (sPindle), 5 rpm, or equivalent, and the 'viscosity of the composition is made from the composition and determined. After the stability can be measured, generally * after the composition is made, at 25 ° C ± rC and the pressure of the environment for at least 24 hours, and then after the rotor rotates 3 sec seconds at a temperature of 25 ° C ± rc The apparent viscosity of the composition was measured. The topical compositions of the present invention are typically formulated to be less than or equal to pH 9.5, and generally range from about ·4·5 to pH 9, preferably from pH 5 to _PH 8.5. In some instances, especially an additional active agent such as salicylic acid, to fully exert the activity of the additional active agent, requires a lower pH, so these compositions are typically formulated to have a pH in the range of about 2.5. From 5 to about 4, preferably from about 2.7 to 4, the topical composition of the present invention may comprise a broad and diverse selection of optional ingredients which are chemically compatible with the physical components and which are not excessively damaged. The stability, efficacy, or other benefits associated with this composition. The optional component can be a dispersed, liquefied or the same material as the carrier of the composition. Examples of optional components of the present invention include softeners, oil absorbents, anti-caries agents, binders, buffering agents, denaturing agents, surface astringents, external analgesics, film formers, wetting agents, sunscreens, and fragrances. Agent, pigment, ♦, skin stabilization and treatment reagents, preservatives, propellants, skin penetration enhancers, solvents, suspending agents, emulsifiers, detergents, thickeners, melting agents, waxes, sunscreen lotions, sun-drying agents, Antioxidants and/or free radical scavengers, chelating agents, anti-acne agents, anti-inflammatory agents, dandruff/release agents, organic hydroxy acids, vitamins, and natural extracts. Described in Harry, s Cosmeiicology, 7th Ed.? Harry &amp; Wilkinson (Hill Publishers, London 1982) ^ Pharmaceutical Dosage

Forms—Disperse Systems; Lieberman, Rieger &amp; Banker, Vols· 1 (1988) &amp; 2 (1989); Marcel Decker, Inc.; The Chemistry and Manufacture of Cosmetics, 2nd· Ed·, deNavarre (Van Nostrand 1962- 1965); and the examples of the substance in The Handbook of Cosmetic Science and Technology? 1st Ed. Knowlton &amp; Pearce (Elsevier 1993) can be used in the present invention. The topical compositions of the present invention are prepared by conventional methods for making topical compositions, and typical methods include mixing the materials into a single state, with or without heating, cooling, vacuum, and the like, in one or more steps. . 42 200808973 The localized crude product of the present invention can be used to treat or alleviate the onset of injury or attack on the skin, and can also be used to control or improve skin condition. Whereas the use of the topical composition of the present invention is a topical administration of the composition to the skin in a safe and effective amount, and the dosage, the frequency of administration and the period of use of the composition are based on the amount of activity of the composition administered. The standard quantities of the pre- and period periods are quite different. ·

I &lt; The range of compositions of the present invention is beneficial to the appearance and/or feel of the skin. Typical compositions are used in an amount of from about 0.1 mg/cm to about i mg/cm2', such as 2 mg/cm2. In general, a composition can be used r times a day, and the frequency of application can be increased from about once a week to about three times a day or multiple times. The topical composition of the present invention can provide an obvious improvement in skin discomfort immediately after administration, which immediately improves the thief's discontinuity of covering or covering the skin (including those related to skin aging, such as The pores become larger, or even provide - the color or color of the skin. The compositions of the present invention may also be provided after prolonged topical application - the apparent improvement in skin navigation - the fine-grained application includes the long-lasting, sustained topical use of the composition, preferably at least about one week between (4) , -month, three months, six months, or one year. Long-term control of skin conditions involves an improvement in skin condition after multiple topical administrations. A preferred method for controlling the skin condition is to take a skin lotion, a medicinal milk taste, a beauty product, or a composition in the form of a reversal, and the duty will stay in the skin red (four) time to _ some Wei, prevention Disease, 200808973: The benefits of his treatment (ie "leave, ^

:' 1 composition does not contain cleansing products for cleansing. At J i2 hours. Λ ^, 3G minutes, 1 error, or greater than about the efficacy of the present invention or the composition can be evaluated by in vitro (5) rushing = body coffee) experiments, for example, can be performed in vitro '靡_帽_卩_ granule translocation due to performance or activity T force corpus callosum. In the test 'can be injected into the human body ^ ^), you can get the effect of the inhibitor on the T cell tuning disorder . According to the sincerity of the narration, the appropriate _ 践 ^ ^ί. . The result is that the suppression of the interpretation of Murakami to other diseases related to abnormal disease, gene expression or activity is abnormally high. Whether a body needs to be treated with a medicinal technique can be formulated by a method known to those skilled in the art. The serotonin of the individual sample is expressed or active in the individual sample. If the multi-credit gene expression or activity in the individual is higher than the amount or activity in the sample taken from the -f-fone, the system is a candidate for treatment with a useful-effective dose of a granulysin inhibitor . The following examples are only intended to illustrate the invention, and are not intended to limit the scope of the disclosure of the present invention, which is believed to be in the scope of the invention. The invention is utilized without departing from the spirit and scope of the invention. All of the referenced (four) readings in this specification are included in the present invention. Examples Clinical samples *

I vesicle cells and blister fluids are taken from the Taiwanese Chang Gung Memorial Hospital, which has the history of the fascinating face group and the toxic table of the dysentery and burns. The patient, the fortune, the diagnosis of the secret bed and the histopathology Known methods such as the discovery of learning _ (see Rcmjeau Invest

Dermat〇U994 Jun; 102(6): 28S_30S). The vesicular cell line used to analyze the amount of expression was taken immediately from the skin tissue diagnosed as Steven's Johnson & Johnson syndrome/toxic epidermal necrolysis tissue, and the blister fluid was taken from many puncture vesicles. In addition, skin tissue sections of five patients with drug-induced plaque papules (MPE) were obtained. The white blood cells in the sputum were obtained from 10 healthy individuals, and all of them had Steven's Johnson syndrome/toxic epidermal necrolysis. In patients with disease or plaque pimples. The immunophenotype of cells in the blister fluid

The type of cells in the vesicles was obtained by flow cytometry. The antibodies and reagents used in immunostaining of vesicles were described below and purchased from BD.

Biosciences anti-CD3, _CD4, -CD8, _CD20 and -CD56 monoclonal antibodies. The phenotypic analysis was performed using defrosted vesicular cells, and after adding GolgiStop (BD Biosciences) in the cell culture medium, 200808973 BD Cytofix/Cytoperm solution was mixed for 20 minutes under the mixture and in a physiological saline solution containing 10% human serum. (PBS), oral culture' followed by staining with a significant immunofluorescent monoclonal antibody such as anti-CD3, -CD4, _CD8, -CD20 and _CD56 f strain antibodies at 4 °C: 20 y. The lion l transfer strain antibody (10) 丨; [) is taken from Yu Na [International Co., Ltd., ^ makes the granulysin in the cells appear, first use the anti-granulysin antibody (RBI; MBL) in the cell 4 The staining was carried out for 1 hour at C, followed by incubation with a secondary FITC_& pE_conjugated anti-mouse monoclonal antibody for 1 hour at 4 °C. Determination of cells in blisters and vesicles. For the analysis of cytotoxicity, the vesicle cells from patients with Stellen's Johnson & Johnson syndrome/sputum epidermal necrolysis are washed and autologous with type IV humans. The herpesvirus-transformed B cells (EBV-transformed B cells) were co-cultured at 37 ° C for 4 hours, and the cells were washed with FITC-labeled anti-CD20 monoclonal antibody and !&gt;E-conjugated flat-hearted sputum ( BD Biosdences) stained for 20 minutes at 4 °C. In order to determine the cytotoxic activity of the vesicular fluid, the present invention uses a standard keratinocyte strain (CCD ll〇6KERTr) to perform the experiment, and the cells are seeded (see(je(|) to a 12-well plate, followed by different concentrations ( 0%, P/〇, 1〇%, and 50%) of the blister fluid was cultured at 37 ° C for 24 hours. After the target cells were incubated with the blister fluid, the target cells were treated with annexin-V at 25. (: After staining for 20 minutes, the keratinocytes of the annexin-V positive reaction were determined by flow cytometry, and the viability of the cells was 46 200808973

A modified version of the tetrazolyl azozolium salt reduction test method proposed by Mosmann (eight (four) deduction with c dew from Res · should be 8 Feb 1:48 (3): 589-^), the result The percentage of the control culture group was expressed, and the cell death was measured using a trypan blue exclusion test. Collapse: array analysis experiment

Extracted from the blister cells or peripheral jk mononuclear cell mononuclear cel PBMC), and then used the Hudn Genome U133 Plus 2.0 array wafer for microarray analysis, and the wafer system included more than 47,000 transcripts. The American Quality Assurance and Control Department was evaluated by the Affymetrix Statistical S5 software. The analysis of the data used the Genedata expressionist and set loooo as the median reference value. To evaluate high-yield genes, the maximum p-value was set to 0·02, and T-test was used to analyze the different expressions between the two groups. It was found that 200 genes had significant correlation (P value). &lt;4.1x10 4) 'The cluster performance of the 200 genes was further analyzed. Quantitative real-time reverse transcription polymerization, the production of P. sinensis, and the preparation of human vascular cells from human tissues and cultured vascular cells. Inversion 47 200808973 Recorded polymerase chain reaction, using the Master SYBR Green I kit, the amount of transcript was determined by LightCycler instrument (R0Che Molecular Biochemicals) (see Shichiri ei a/. J Biol Chem. 2001 Nov 9; 276 (45) :41998-2002; Shichiri et aL Cancer Res. 2002 Jan 1;62(1):13-7), the absolute copy number is determined using conventional methods (see Matsushita β fl/· Br J Haematol· 2001 • Mar; 112(4): 916_26). · RNA is also isolated from peripheral blood mononuclear cells or vesicular cells

• Yes, quantitative real-time reverse transcription polymerase chain reaction system using SYBR

Green I ( Rodhe Applied Science ) measures the amount of transcripts using a LightCycler instrument using the following oligonucleotides: granule lysin · 5,-TCTCTCGTCTGAGCCC-3, 5,-GCAGCATTGGAAACACT-3, perforin · 5,-ACCAGCAATGTGCATGTGTCTGTG_3 , 5,-GAAGGA GGCCGTCATCTTGTGCTT-3, φ granzyme-Β : 5,-TGCAGGAAGATCGAAAGTGCG-3, 5, GAGGCATGCCATTGTTTCGTC-3,

Fas ligand: 5, TCATGGTTCTGGTTGCCTTG-3, 5,-AAATGGGCCACTTTCCTCAG-3, /3-actin: 5,-ACATCCGCAAAGACCT-3, 5,-AGGG TGTAACGCAACTA-3, 48 200808973 Sodium decyl sulfate polyamine Steroidal electrophoresis (SDS-PAGE) 舆Immunotransfer method (immunoblottii^ Dissolve tissue or cell samples in a reduced sample buffer containing 60 mM trihydroxyethylamine stearate-hydrochloride at pH 6.8 Acid (Tris-H.Cl), 2% (W/V) sodium sulfonate sulfate (SDS), glycerin of Qing (4), and 50 thiothreonose-alcohol (dithiothreitoh DTT), the sample was separated by 15% sodium dodecyl sulfate sodium polyacrylamide gel electrophoresis, and the pre-stained standard molecular weight protein was used as the calibration standard for egg drug movement in advance, and then semi-dry (semi) -dry) Transfer device in 10 mM cyclohexylaminopropanesulfonic acid (CAPS) containing 10,4 (v/v) sterol and 〇·〇5°/0 (w/v) In the transfer buffer of thiothreitol, and transferred at a current density of 1 mA/cm 2 for 30 minutes to transfer the protein in the colloid On the polyvinylidene difluoride membrane (PVDF membrane), and after the transfer is completed, the membrane is placed in a blocking buffer to block non-specific binding, the blocking buffer Tris buffered saline (TBS) with TBST (containing 〇.1% 〇/¥) polyoxyethylene sorbitan monolaurate (Tween-20) ρΗ7·5 And 5% (w/v) skim milk prepared from skim milk powder, followed by rabbit anti-jk clear (rabbitantisera) and peroxidase-labeled horse anti-rabbit secondary antibody, respectively. Both antibodies were appropriately diluted with blocking buffer, and then the excess antibody was washed away with TBST, and then the imaging reaction was performed using Western 200808973 square ink dot deduction agent (ECL or ECL_plus, Amersham). The film is sensitive. The radiolabeled protein on the gel was fixed with Amplify (Amersham), dried, and sensitized at _70 °C according to the manufacturer's instructions. ' 教 签 之 之 之 ( ELISA — — — — ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ The buffer solution (0.1% polyoxyethylene sorbitan monolaurate phosphate physiological diet • saline) blocks non-specific binding and is continuously reacted for 2 hours at room temperature in blocking buffer; Reaction in blocking buffer containing 〇.l//g/ml phytochemicalized RC8 monoclonal antibody for 1 hour; and anti-biotin streptococci containing 〇.〇5u/ml /3 -galactosidase Washing buffer of zebra-galactosidase_conjugated streptavidin. The plastic well plate was rinsed with a rinse buffer between the two reactions, and the well plate was prepared to contain 0. 02% bovine serum albumin, _100 mM sodium chloride (NaCl), and 1 mM magnesium chloride (MgCl2). 1 OmM phosphate buffer (ΡΗ7·0) in 4.4mM 4_mercapto- umbelliferone-g-D_ galactose "·methylumbelliferyl-gD-galactoside" at 37 C for 17 hours' followed by lOOmM glycine - Glycine hydroxide (pH 10.3) terminates the enzyme reaction, and a fluorescence meter (Cyto-Fluor 4000 fluorescence multi_well plate reader, Applied Biosystems) is used to measure the fluorescence intensity at a wavelength of 360 nm and 460 nm for each of the excitation and emission light. 50 200808973 Immunohistochemical staining method The skin samples are taken from patients with Steven's Johnson & Johnson syndrome/toxic epidermal necrolysis and plaque-like ablation, and these skin samples are co-cultured with specific antibodies. Anti-, granulysin antibody (RC8, MBL, Japan), anti-granzyme: B antibody (abeam, UK), anti-perforation antibody (Kamiya, USA), and anti-Fa§ ligand antibody (]^1^ 4,

Alexis, Switzerland). Recombinant functional granulysin performance 舆 Purification 蚤, full-length granulysin cDNA (encoded as guanidine rim 1 to arginine 136) cloned (ci〇ned) into pcDNA-TA vector (Invitr〇gen), and transformed (transformation) to E. coli BL21 (DE3), the E. coli can be expressed as a C-terminal (histidine) 6-labeled recombinant 15 kDa granulysin; and with a control vector containing no full-length granulysin And purification comparison. The expression of the recombinant protein was confirmed by Western blotting method, and the recombinant granulysin was purified by using an affinity column (nickelc〇lumn) in a denatured state, and polymerized with 15 〇/〇 of sodium lauryl sulfate. The acrylamide colloid is further purified by purifying the protein with a molecular weight of 15 kDa. After purification, the protein is reduced by the thiol alcohol, even if the protein is refolded in the presence of oxidized dithiothreitol (ref〇 Ld), and dialyzed against triammonium sulphate stearate, then the histidine label was removed, and a protein quantitative assay (c〇omassie blue protein) was used. Assay) The protein was quantified based on serum albumin (albumin), and the total protein concentration prepared was consistent with the concentration of granulysin measured by enzyme immunoassay, and the purity of the protein was 150/. The Coomassie staining results for the sodium dodecyl sulfate polyacrylamide gel were 95% consistent, and the purified Fas ligand, perforin, and granzyme_B protein were purchased from Alexis, Switzerland. .

I. Results of the St. Johnson's Johnson & Johnson syndrome/toxic epidermal necrolysis cytotoxicity in the vesicles of the cytotoxic CD8 positive T and natural killer fine face ♦ i measured from five people with Steven's Johnson & Johnson syndrome / toxicity The immunophenotype of cells in the vesicular fluid of patients with epidermal necrolysis, the drug-induced epilepsy (carbamazepine; cases 1, 4, and 5), diphenytoin (case 2), And amoxicillin (case 3), regardless of the induced drug, the main cell line in the vesicular fluid CD56-positive natural killer cells (48% to 100%) and CD8 positive antigens expressing CD8 positive subgroups T cells (22% to 70%) or CD56-positive natural killer T cells, CD8-positive CD56-positive cell lines have predominantly increased natural killer cell subtypes (4% to 44%) (see Table 1). Table 1. Stroke phenotype analysis of Stellen's Johnson & Johnson syndrome/toxic epidermal necrolysis disease 200608973 Case 1 (AdrD791) Phenotype/inducing drug SJS-TEN/ Epileptic long-lasting film-coated ingot case 2 (AdrD811) SJS -TEN/ Diphenyltestine Case 3 (AdrD835) 'rm/^ Amoxicillin Case 4 (AdrD826) SJS-TEN/ Epilepsy Long-acting Membrane Key Case 5 (AdrD7〇9) Epilepsy

CD4+?CD56+ CD8+,CD56+ (ΝΚΊ) 〇% 4% 0% 0% 44% 33% 8% 22%

The cytotoxic activity of vesicular cells on the cells of autologous target cells to kill the blister cells was determined by transfecting vesicular cells (acting cells; sample number 3) and human herpesvirus (EBV) as target cells into autologous B. The cells (without autologous keratinocytes) were co-cultured for four hours, and evaluated by annexin-fluorescein isothiocyanate (annexin-FITC) before apoptosis, and found to be in the absence of vesicular cells. In the case where the dead cell's 'relative' ratio of the acting cell/target cell was 5 to 1, the vesicular cells induced 31% of B cell apoptosis, and the additional drug was only slightly increased to 37%. The results are summarized in the three patients in Table 2 below. Table 2, cytotoxic activity of vesicular cells 53 200808973 Case 1 Case 2 Case 5 Autologous B cells 44% 32% 31% Autologous B cells + induced drugs 46% 30% 37% As shown in Table 2, all vesicular cells are self-contained B cells transformed by EBV have a strong cytotoxic effect, and the addition of induced drugs can not significantly increase the cytotoxicity, indicating that the vesicle cells have been activated. Induced by vesicles! Apoptosis and sclerosing phenomenon of keratinocytes 1 The toxicity of vesicular fluid to keratinocytes (the target skin cells in patients with Stellen's Johnson syndrome/toxic epidermal necrolysis) By culturing keratinocytes (KER-tr) in 1%, 10% of the acute phase (less than 3 days) of the Stevenson Johnson syndrome (sample number 4) or toxic skin necrosis (sample number 5), Or 50% blister fluid in the culture solution for 24 hours. The blister fluid of scald injury was taken as the control group with 50% concentration. The results are shown in Table 3 below: cytotoxic activity of vesicular cells on keratinocytes _ cell viability _ _% blister fluid 100% 1 〇 / 〇 blister liquid ~ 98% Occupational water bubble ~90% 50% blisters ~72% -^ -98% As shown in Table 2, 50% and 10% of the blister fluid in the culture medium induces cells of the keratinocyte strain. It is positively correlated with the concentration of the blister fluid. 54 200808973 尤 涂 _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ The overall gene expression profile of the vesicular cells, which is derived from the peripheral blood mononuclear cells compared to the map of the peripheral blood mononuclear fine riding of the patients with the Stable Johnson syndrome/toxic epidermal necrolysis (sample number 6) The comparison is due to the presence of cells in the skin blister fluid (IV), which are present in peripheral monocytes. On the ride, there are patients who are not connected, and are in any active period of medical treatment (3 days of this disease), and each group, gene sputum (Ul33plus2.〇, Affymetrix) · Analysis of the completion of the use of each patient's RNA sample. * ▲ It was found that there were 2 genes with different performances, which were used for statistical analysis. The result was that the 200 genes with different expressions were detected and tested in St. Johnson's Johnson & Johnson syndrome/toxicity. Peripheral blood mononuclear cells of epidermal necrolysis, found that there are significant upward basal sputum in vesicular cells, and most of the up-regulation _ genes are related to immune and cellular: t-killing 自然/natural killer cell pathways, including Granule lysin, 4 enzyme = CD3 antigen, cathepsin B (c base such as b), tissue: white enzyme L, and complement lq (deleted coffee, add). Interesting in the cytotoxic refractory egg, the transcription of granulysin: showing a dramatic increase in transcripts and higher than the transcriptional production of granzyme B and perforin, as the result is confirmed by an instant polymerase chain reaction, and The up-regulated genes and relative p-values are listed in Table 4 below. 55 200808973 Table 4: Up-regulated genes in vesicular cells

• To confirm the results of this preliminary screening, the use of real-time quantitative polymerase linkage 4 should be evaluated for _ eight expressions of granulysin, particulate gas beta, perforin, and Fas/I^s ligands in vesicular cells. Comparison of relative performance of peripheral blood mononuclear cells from peripheral blood mononuclear cells taken from Steven's strong syndrome/toxic epidermal necrolysis (sample 13) or from healthy individuals (sample 8) The results showed that the mRNA expression of granulysin in the vesicular cells of the venom of the venom of the dysentery group was higher than that of the Fas ligand, perforin, granule B, and granule B. And the amount of granulysin transcripts in the vesicles in the patients with Stellen's Johnson & Johnson/toxic epidermal necrolysis is 17.1 times that of the peripheral blood mononuclear cells, and the same day is also for healthy individuals. 12 times in peripheral blood mononuclear cells; in addition, it was also found that the transcription product 1 of Fas ligand, perforin' or granzyme Β in vesicular cells of patients with Stables syndrome/toxic epidermal necrolysis is Patient periphery 9.9, 3·9 and 1 times of the transcripts in liquid monocytes, and also 5.9%, 3.4 and 3.2 times in the surrounding menopausal monocytes of healthy individuals, but the increase of the three The amount is low 56 200808973 The increase in granulysin transcripts. The question of the label &amp; prime protein resilience is manifested in the St. Johnson Johnson & Johnson syndrome / Maoba: &amp; skin lesions in patients with sputum sputum plaques to further confirm the results of the aforementioned niRNA, 遂 detection of the history of the Johnson & Johnson syndrome / Granulysin protein in skin lesions in patients with toxic epidermal necrolysis, using immunohistochemical staining to observe Steven's Johnson & Johnson syndrome/toxic epidermal necrolysis (sample 5) or plaque (Sample 3) The skin section of the patient was found to have a large amount of keratinocyte necrosis, and the immunohistochemical staining analysis showed that the Stigzil Johnson Syndrome/Toxic Epidermal Necrolysis Table. Around the necrotic area of the skin exfoliation A large amount of granulysin is stained, and only a small amount of granulysin is stained around the necrotic area of the flaky papules. In addition, other cytotoxic proteins (perforin, granzyme B, and Fas ligand protein) were found to be low in the samples of the Stellen Johnson syndrome/toxic epidermal necrolysis granules. Performance. Then, using granulysin and anti-CD antibody, flow cytometry-double staining was performed on the vesicular cells. It was found that granule lysin was mainly expressed in CD8-positive and natural killer T cells. Shichawen Johnson & Johnson Syndrome / Toxicity Table 高 鬏 之 之 之 之 之 高 高 高 高 高 高 57 57 57 57 57 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 The blister liquid of the necrolysis granules, the granules of the lysin, the expression of the f, and the Western blotting method of the present invention, the results show that the blister fluid of the patient of the dynasty syndrome/toxic epidermal necrolysis is not _ The granules of the molecular weight 9kDa type dissolve the cap protein, but can reach a large amount of granule lysin protein with a molecular weight of 15 · difficult type, and the blister liquid towel 贞 ] γ 贞 of the patient suffering from severe burn can not detect any granulysin protein . Then, the enzyme-linked immunosorbent assay measures the concentration of granulysin and other cytotoxic proteins in the vesicular fluid of the patients with dysplasia, dysplasia, and dysplasia, and then burns or tianhe (bullGuspemphig()id) silk was used as a test group. It was found that there was a higher concentration of lion lysin in the vesicular fluid of patients with a history of strong disease scale/toxicity. The concentration value was 6920.61 ng/ml (633.33). To 63392 3 Bu sample number 29), the relative 'burning and class Tianhe patients' water suture lysine has a lower concentration, the concentration value is 55.58 ng / ml (sample number (5) and 22 · 07 ng /ml (sample number 5). The highest average granulysin protein expression in vesicular fluid is found in toxic epidermal necrolysis (more than 30% body surface area ^ is in the case of Steven's Johnson & Johnson syndrome and toxic epidermal necrosis Symptoms (more than 29% of body surface area) and Steven's strong group (less than 10% of body surface area)., 58 200808973 Turning to the illusion - _ New Zealand view of the lysis of the egg Face-to-face increase, Fas ligand, perforin, and The egg of the enzyme B is from the lower eye, the phase is soluble Fa (four) body: 0.41 n_ (〇 to 2.43, sample number 29); perforin: , i^ng/mKO.oobuw, sample number 29); granzyme b: 〇96 • η__4_.36' sample number 29), and all three were not significantly associated with clinical severity. · In vitro - wYm, cytotoxicity test I! ------ « • ^ to explore the cytotoxic effects of various cytotoxic proteins, this, the invention uses keratinocyte cells as target cells, in vitro test results found 'Complementary 15kDa granule lysin is significantly cytotoxic at concentrations present in the vesicular fluid of Steven's Johnson & Johnson syndrome/toxic epidermal necrolysis (32% cell death at 400 ng/ml), notably, Only the granulysin is present in the Steining Johnson Syndrome syndrome table. The concentration of the necrosis _ 丝丝的水巾 towel has the cytotoxicity of lin, Fas ligand, perforin, and granzyme B are present in the St. Johnson Johnson & Johnson syndrome/ The concentration in the vesicular fluid of patients with toxic epidermal necrolysis is not cytotoxic (concentration is about 1 ng/ml). The direct injection of granule lysin by Youlu will be responsible for the sputum necrosis of sputum to explore the effect of granulysin in vivo (plus v/v 〇), and the purified complement of concentration of 4000 ng/ml 15]^^ Granulysin directly 59 200808973 was injected into nude mice via skin injection, and the phosphate physiological saline solution and the carrier containing no granules; the solution of glutathione (VeCt〇r) was separately injected into the nude mouse body. As a control group, after 5 injections in 5 hours (1 hour injection), significant skin necrosis was found at the injection site. Conversely, the same phenomenon was not found at the injection site of the control group. - * 1 by uranium The data showed that 'vesicular cells f are mainly CD56-positive and CD8-positive T cells, and the vesicular fluid is present in keratinocytes (the target skin cells in the vestige-strong syndrome/toxic epidermal necrolysis)

• Significant cytotoxic activity. In addition, the vesicle cells are cytotoxic regardless of the presence or absence of the induced drug, thus indicating that the vesicular cell line has been activated. However, this result is different from the results published by Nassif et al. in 2002. (Nassif, j. AUergy elijL

Immunol·2004N〇V;114(5):1209_15), Nassif et al. found that vesicular cells were cytotoxic only in the presence of drugs, and the degree of cell toxicity (6-12%) found by Nassif et al. was lower than this. The degree of cell toxicity found in the hair • (3〇_46%). The results confirmed for the first time that secretory _ granulysin is a key molecule leading to diffuse keratinocytes and that granulysin is a key link in the pathogenesis of Shi Disheng syndrome/toxic epidermal dysfunction. According to the present invention, other toxic proteins, such as granzyme B, perforin, or soluble Fas ligand, in vesicular fluid, have been found to have a significant correlation with the onset of sensitization syndrome/toxic specticulosis. The high performance of granulysin is 200,808,973. It is twice as large as the other toxic proteins f, and it can be found in vitro and in vivo that the presence of _ lysosol concentration in the vesicle has significant cytotoxicity. In the past, the study of granulysin mainly focused on the age of 9kDa mature particles, that is, the modification of the precursor before the transfer (see Krensky as α/· Am J Trai|splant· 2005

Aug; 5(8): 1789·92). The granulysin is known as a cation-bearing molecule and is present in the granules of human lymphocytes and natural killer cells. In contrast, the 9kDa granule lysin is similar to other cell-dissolved proteins i. The calcium-dependent pathway Cealcimi Miefjendentpathway) secretes to the intercellular space between the acting cells and the target cells, and the 9kDa granulysin can bind to the surface of the target cell by charge to generate ion mobility phenomenon, resulting in a nerve sheath. Activation of sphigomylinase leads to the production of ceramide (see Gamen ea/J. Immunol. 1998 Aug 15;161(4)·1758_64), so 9kDa granulysin has a variety of microorganisms And the cytolysis activity of tumors (see Krensky as α/·, Am·J·Transplant· 2005 Aug; 5(8): 1789-92), and simultaneous ion movement phenomena can also cause damage to granulocytes, resulting in cytochromes C (cytochrom C) and apoptosis inducing factor release leads to stylized cell death (see pard〇, fl/., J. Immunol. 2001 Aug 1; 167(3): 1222_ 9). In addition, studies have also indicated in the past that granulysin is a chemotactic and pro-inflammatory inflammatory activator (see Deng β a/·, J Immunol· 2005 May 61 200808973 1; 174(9): 5243-8) The present invention utilizes two antibodies (multiple antibodies and monoclonal antibodies (Standford)) which can recognize two different 15kDa type and 9kDa mature type granulysin. However, the present invention detects in a blister fluid. The granulysin was obviously 15kDa type, and no 9kDa mature granule lysin was detected. The 15kDa granule lysin, a precursor of the 9kDa type, was confirmed to be secreted by natural killers and tau cells via a non-granule exocytotic pathway, and the tau cells were co-cultured with gas-labeled cells. At the time, the amount of granulysin exhibited increased JE appearance. ♦ E· ♦ The foregoing results show that the 15kDa type granulysin alone has effective cytotoxicity. The purified 15kDa granule lysin was injected into the skin of the mouse by injection, and the concentration of the granulysin present in the vesicular fluid of the Stellen's Johnson & Johnson syndrome/toxic epidermal necrolysis septic solution was injected. Causes significant necrosis of the epidermis and dermis, showing that the 15kDa granule lysin is not a non-specific and untreated product' and the necrosis or blistering needles of the Stellen's Johnson & Johnson syndrome/toxic epidermal necrolysis The high concentration of 15kDa granules secreted extracellularly will rapidly lead to the growth/necrosis of the sulphate cells. This result explains the histopathology of the Stellen's Johnson & Johnson syndrome/toxic epidermal necrolysis. It is often found to cause extensive epidermis. A small amount of necrotic dermal layer mononuclear ball / / / 闰 (Quinn et aL, Dermatol. 2005

Jun;141(6):683-7) 〇62 ^υ〇8〇8973 The expression of granulysin in vesicular fluid is significantly correlated with clinical severity, so toxic epidermal necrolysis has the most extensive skin invasion. Phenomenon (greater than 30% body surface area), the highest amount of granulysin, followed by the Stevenson Johnson Syndrome and Toxic Epidermal Necrolysis Symptoms and the Steven Johnson Johnson Syndrome, showing that granulysin can be used to monitor The progression of these diseases, as well as the new treatments used to develop the above-mentioned lethal diseases with high morbidity and mortality. In addition, it has also been found that the increase in granulysin is only found in the St. Johnson syndrome/toxicity table, Zhao Congjie, and there is no increase in other water-derived skin diseases. It is shown that measuring the granule lysin helps to check the same water &lt; skin _, which is free from skin sectioning. It is obvious that the study has been more difficult than the Stevens Johnson & Johnson syndrome/toxic epidermis. First, transplanting the skin against the remaining disease or "the performance of his internal organs are similar to the Stevenson Johnson syndrome / toxic ^ epidermis bad hearing disorder" can be found in the intestines, liver and transplanted host disease Skin; in addition, according to the above results, it should be re-examined the main pore copper peak b method of cell regulation regulated by the current emphasis; further, the result of the present invention is the poisoning of the target cells regulated by T cells. The role of the hand cell / poisonous tau cell direct contact with the target cell is proposed different views. The characteristics of the households disclosed in this specification can be combined freely, and the disclosure of 63 808973 can be equivalent or equivalent change The invention is exemplified only to clarify the scope of the present invention. The sub-Asian and the non-Asian are used to define the scope of the present invention. According to the above, any skilled person can easily understand the present invention. The hair of the present invention is: and without departing from the spirit and scope of the present invention, it is possible to make appropriate adjustments and accessories according to various uses and conditions. Therefore, the scope of protection of the present invention is attached. The definition of the scope of the application patent shall prevail. 64 200808973 [Simple description of the diagram] None [Main component symbol description]

Claims (1)

200808973 X. Scope of Patent Application··1.------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------ And the amount of the conjugated silk and the value of the lysin, if the amount of lysin is higher than the preset value, thereby determining that the sputum individual has granulysin-regulated autoimmune disorder or has 罹® Risks </ RTI> 2, as described in the patent application 4 | Item 1, wherein the autoimmune dysfunction is Stevens-Johnson syndrome 'SJS, toxic epidermal necrolysis ( T〇xic epidermal necrolysis 'TEN), transplantation against host disease, Beth's disease, ankylosing spondylitis, systemic lupus erythematosus, dermatomyositis, polymyositis, and organ transplant rejection. 3. The method of claim 1, wherein the test sample is a one-piece liquid sample. 4. The method of claim 3, wherein the body fluid sample is a blister night sample or a serum sample. 5. The method of claim 1, wherein the granulysin comprises the sequence of SEQ ID NO: 1 or a fragment of the sequence. 66. The method of claim 1, wherein the granulysin comprises the sequence of SEQ ID NO: 3 or a fragment of the sequence. 7. The method of claim 1, wherein the granule lysin comprises the sequence of SEQ ID NO: 5 or a fragment of the sequence. i &lt; 8, a method for detecting an autoimmune dysregulation process regulated by granulysin in vivo, comprising measuring the amount of granule lysin taken from one of the individual samples. _ As claimed in the method of claim 8, the test sample is a one-piece liquid sample. ♦ ♦ 1〇, as described in the scope of claim 9 of the patent application, wherein the body fluid sample is a blister sample or a serum sample. 11. The method according to claim 8, wherein the autoimmune disorder is Steven's Johnson & Johnson syndrome, toxic epidermal necrolysis, transplantation versus host disease, Beth's disease, and rigid spine. Inflammation, systemic lupus erythematosus, dermatomyositis, polymyositis, and organ transplant rejection. 12. A method of assessing a prognosis of a subject, the subject planning to receive or have received a medical treatment or organ transplant, the method comprising determining the amount of granulysin present in a test sample taken from the individual. The method of claim 12, further comprising administering to the individual a therapeutic drug or transplant prior to obtaining the individual sample. 67 13 200808973 14 If you apply for the scope of the patent before measuring the amount of performance, touch. The method of 12, further comprising the step of attaching the sample to a therapeutic drug or a transplant. For example, the 12th patent application scope is a one-piece liquid sample. The method of the item, wherein the test sample 16 17 v , 18, wherein the body fluid is a blister sample or a serum sample, as described in claim 15 &lt;RTIgt; A method for treating an autoimmune disorder regulated by granulysin, which comprises an effective granule lysin inhibitor required for administration. For example, the method described in Item 17 of the full-time application, wherein the inhibitor is an antibody that specifically binds to granulysin. 19. If the method described in claim 17 (4), the inhibitor is an RNA molecule. 20. A method for treating a granulysin-regulated autoimmune disorder comprising: administering a test composition to a cell expressing granulysin; and determining the presence of the test composition. Or the amount of intracellular granulysin present, if the amount of intracellular granulysin present in the test composition is lower than the cells in the absence of the test composition, thereby indicating that the test composition can be used to treat the granules dissolved Immunoregulatory autoimmune disorders. 68 200808973 21. The method described in claim 2G of the patent scope, wherein the disorder is a strong syndrome of toxicosis, toxic epidermal necrolysis, transplantation, main disease, Bezi's disease, ankylosing spondylitis, Systematic Lupus erythematosus, dermatomyositis, polymyositis, and organ transplant rejection. 22, a method for treating an autoimmune disorder in which a test compound is controlled by an intimacy, comprising: * providing a multi-peptide containing a granulysin sequence; 'utilizing &quot; a multi-peptide and a test combination The molecular mimetic turn; and the debt fl flanked by the composition of the intermolecular _ knot, f the test composition can be linked with the neopeptide, thereby determining that the test combination is an autoimmune disorder that can be transfused with refractory Candidate 23, = the method described in the scope of claim 22, wherein the disorder is a strong genus of hernia, her epidermal necrolysis, transplantation of several major diseases, Bezi's disease, ankylosing spondylitis, Systemic lupus erythematosus, dermatomyositis, polymyositis, and organ transplants 69 200808973 VII. Designation of representative figures (1) The representative representative of the case is: (No). (2) A brief description of the symbol of the representative figure: None 8. If there is a chemical formula in this case, please disclose the chemical formula that best shows the characteristics of the invention: jjIL t f