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WO2003054231A1 - Méthode d'examen de maladie allergique - Google Patents

Méthode d'examen de maladie allergique Download PDF

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Publication number
WO2003054231A1
WO2003054231A1 PCT/JP2002/013071 JP0213071W WO03054231A1 WO 2003054231 A1 WO2003054231 A1 WO 2003054231A1 JP 0213071 W JP0213071 W JP 0213071W WO 03054231 A1 WO03054231 A1 WO 03054231A1
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WO
WIPO (PCT)
Prior art keywords
gene
gbp1
indicator gene
indicator
allergen
Prior art date
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PCT/JP2002/013071
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English (en)
Japanese (ja)
Inventor
Naoko Nagata
Nei Yoshida
Tadahiro Oshida
Ryoichi Hashida
Yuji Sugita
Gozoh Tsujimoto
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Genox Research Inc
National Center for Child Health and Development
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Genox Research Inc
National Center for Child Health and Development
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Priority to JP2003554931A priority Critical patent/JPWO2003054231A1/ja
Priority to AU2002354228A priority patent/AU2002354228A1/en
Publication of WO2003054231A1 publication Critical patent/WO2003054231A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/08—Antiallergic agents

Definitions

  • the present invention relates to a method for detecting an allergic disease.
  • Allergic diseases are considered to be multifactorial diseases.
  • bronchial asthma ditopic dermatitis is caused by the interaction of the expression of many different genes, and the expression of these individual genes is affected by several environmental factors. For this reason, it was very difficult to elucidate the specific genes that cause allergic diseases.
  • Allergic diseases are thought to involve the expression of genes with mutations or deficiencies, or overexpression or reduced expression of specific genes. To understand the role of gene expression in disease, it is necessary to understand how genes are involved in pathogenesis and how external stimuli, such as drugs, alter gene expression.
  • allergen-specific I g E measurement, leukocyte histamine release test, or lymphocyte blastogenesis test, and the like.
  • the presence of allergen-specific IgE is evidence of an allergic reaction to the allergen.
  • some patients may not always be able to detect allergen-specific IgE.
  • diagnosis Testing must be performed for all required allergens.
  • Leukocyte histamine release test ⁇ Lymphocyte blastogenesis test is a method for observing the response of the immune system to allergens in vitro. These methods are complicated in operation. On the other hand, a method for utilizing the immune response observed when a patient is actually brought into contact with an allergen to diagnose allergens (the latter) is also known. Prick 'tests, scratch' tests, patch 'tests, intradermal reactions, or provocation tests are included in this type of test. While these tests can directly diagnose a patient's allergic reaction, they can be described as highly invasive tests that actually expose subjects to allergens.
  • test methods have been attempted to prove the involvement of allergic reactions regardless of the allergen.
  • a high serum IgE level may indicate that the patient has an allergic reaction.
  • the serum IgE value is information corresponding to the total amount of allergen-specific IgE. Although it is easy to determine the total amount of IgE regardless of the type of allergen, IgE may be low in patients with diseases such as non-atopic bronchitis.
  • markers for allergic diseases could be provided, which would have minimal invasiveness to patients and could easily obtain information necessary for diagnosis. Since such markers are thought to be deeply involved in the development of allergic diseases, they may be important targets not only in diagnosis but also in control of allergic symptoms. Disclosure of the invention
  • An object of the present invention is to provide an indicator gene that enables detection of an allergic disease. Furthermore, an object of the present invention is to provide a method for testing allergic diseases and a method for screening therapeutic drugs for allergic diseases based on the index.
  • Various blood cells are closely related to the immune response. For example, for mite antigen The resulting immune response proceeds as follows. First, monocytes phagocytose mite antigens and present their degraded products to T cells as antigens. Only T cells that can recognize the presented mite antigen peptide will respond to antigen presentation. T cells that respond to antigen presentation produce various cytokines that determine the direction of the subsequent immune response (allergic reaction).
  • the cytokines produced at this time are cytokines closely related to allergic reactions, such as IL-4, IL-5, and IFN- ⁇ .
  • IL-4 cytokines closely related to allergic reactions
  • IFN- ⁇ IFN- ⁇
  • T cell activity for example, in B cells, interaction with T cells and stimulation with IL-4 initiate the production of mite antigen-specific IgE.
  • the present inventors thought that by observing a change in gene expression in blood cells supporting such an immune response system, it was possible to isolate genes related to the allergic reaction. Based on such a concept, the present applicant has succeeded in isolating the next gene whose expression level is changed in peripheral blood cells of a hay fever patient, and has filed a patent application.
  • Pollinosis-related gene 3 7 3 (WO00 / 65046) Pollinosis-related gene 4 19 (WO00 / 65045) Pollinosis-related gene 5 13 (WO00 / 65049) Pollinosis-related gene 58 1 (WO00 / 65048) Pollinosis Related gene 7 95 (WO00 / 65050) Hay fever-related gene 62 7 (WO00 / 65051) Hay fever-related gene 44 1 (WO00 / 73435) Hay fever-related gene 46 5 (WO00 / 73439) Hay fever-related gene 7 8 7 (WO00 / 73440)
  • genes having a difference in expression level Similarly, in peripheral blood mononuclear cells of patients with allergic diseases, the following genes have been identified as genes having a difference in expression level, and a patent application has been filed. These genes were identified by gene expression analysis with GeneChip.
  • genes are genes whose expression levels are different in cells isolated from peripheral blood. Such expression is called spontaneous expression.
  • PBMC peripheral blood mononuclear cells
  • the present inventors have further improved this method, and searched for a gene whose expression level is changed particularly in T cells and monocytes after allergen treatment.
  • T cells and monocytes are cells that determine the immune response. Therefore, an important role is predicted for genes whose expression levels fluctuate in T cells and monocytes when an allergic response to allergen occurs.
  • the present inventors have revealed that the expression level of the GBP1 gene is significantly reduced with an allergic immune response. Based on these findings, the present inventors have found that it is possible to test for allergic diseases or to screen for therapeutic drugs for allergic diseases using the GBP1 gene as an index, and completed the present invention. That is, the present invention relates to the following methods for testing allergy, therapeutic agents for allergic diseases, screening methods thereof, animal models for allergic diseases, and kits for these methods.
  • a method for testing an allergic disease comprising the following steps, wherein the indicator gene is GBP1.
  • a reagent for testing an allergic disease comprising a polynucleotide containing the nucleotide sequence of the GBP1 gene, or an oligonucleotide having a length of at least 15 bases and having a complementary nucleotide sequence to its complementary strand.
  • An allergic disease test reagent comprising an antibody that recognizes a peptide containing an amino acid sequence encoded by the GBP1 gene.
  • a method for screening a therapeutic agent for an allergic disease comprising the following steps, wherein the indicator gene is GBP1 or a gene functionally equivalent to the GBP1 gene.
  • a method for screening a therapeutic agent for an allergic disease comprising the following steps, wherein the indicator gene is GBP1 or a gene functionally equivalent to the GBP1 gene.
  • Step of selecting a compound that increases the expression level of the indicator gene as compared to the control [11] The method according to [10], comprising a step of stimulating the test animal with an allergen before or after step (1).
  • a method for screening a therapeutic drug for an allergic disease comprising the following steps, wherein the indicator gene is GBP1 or a gene functionally equivalent to the GBP1 gene.
  • a method for screening a therapeutic agent for an allergic disease comprising the following steps, wherein the indicator gene is GBP1 or a gene functionally equivalent to the GBP1 gene.
  • a therapeutic agent for allergic diseases comprising a compound capable of being used as an active ingredient.
  • a therapeutic agent for an allergic disease comprising an indicator gene as a main component, wherein the indicator gene is GBP1 or a gene functionally equivalent to GBP1.
  • a therapeutic agent for allergic monogenic diseases which comprises a protein encoded by an indicator gene as a main component, wherein the indicator gene is the GBP1 gene or a gene functionally equivalent to GBP1.
  • a polynucleotide comprising the nucleotide sequence of the indicator gene, or an oligonucleotide having a length of at least 15 nucleotides having a complementary nucleotide sequence to a complementary strand thereof, and a cell expressing the indicator gene,
  • the indicator gene is the GBP1 gene or a gene functionally equivalent to GBP1.
  • kits for screening a therapeutic drug for an allergic disease comprising an antibody recognizing a peptide consisting of an amino acid sequence encoded by an indicator gene and cells expressing the indicator gene, wherein the indicator gene is GBP1
  • an antibody recognizing a peptide consisting of an amino acid sequence encoded by an indicator gene and cells expressing the indicator gene, wherein the indicator gene is GBP1 A kit that is a gene or a gene functionally equivalent to GBP1.
  • a method for evaluating allergen activity comprising the following steps, wherein the indicator gene is GBP1.
  • Allergen screening comprising the following steps: The method, wherein the indicator gene is GBP1.
  • kits for evaluating allergen activity comprising: 3071
  • the present invention also relates to a method for treating an allergic disease, comprising a step of administering a compound represented by any one of the following (A) to (C).
  • the present invention relates to the use of a compound represented by any one of the following (A) to (C) in the manufacture of a medicament for treating an allergic disease.
  • the present invention relates to a method for producing a model animal of an allergic disease, which comprises a step of reducing the expression intensity of GBP1 or a gene functionally equivalent to GBP1 in peripheral blood mononuclear cells in a non-human vertebrate.
  • the present invention relates to the use of a transgenic non-human vertebrate having reduced expression intensity of GBP1 or a gene functionally equivalent to GBP1 as an animal model for allergic diseases.
  • GBP1 guanylate binding protein-1
  • GBP1 was reported as an antiviral protein induced by interferons (Sylvia L. Aet al. Interferon-induced guanylate burning protein-1 (GBP-1) mediates an antiviral effect against vesicular stomatitis virus and encephalomyocarditis virus Virology 1999 256, 8-14. ⁇ 1 ⁇ 3 ⁇ 41-813 ⁇ 40111 E. et al. Interferon induction of tibroblast proteins with guanylate binding activity. J. Biol. Chem., 1983.258, 7746-7750.).
  • the allergic disease is a general term for diseases associated with the allergic reaction. More specifically, allergens have been identified, and exposure to allergens and the development of lesions are profound! / ⁇ It can be defined that the connection is proved and that the lesion has a proven immunological mechanism.
  • the immunological mechanism means that white blood cells show an immune response by allergen stimulation. Examples of allergens include mite antigens and pollen antigens.
  • allergic diseases can include bronchial asthma, allergic rhinitis, hay fever, or insect allergy.
  • Allergic diathesis is a genetic factor transmitted from a parent with an allergic disease to the child.
  • a familial allergic disease is also called an atopic disease, and the genetically transmitted factor that causes it is atopic predisposition.
  • Asthma is a generic term given to atopic diseases, especially those associated with respiratory symptoms.
  • the method for testing an allergic disease of the present invention includes a step of contacting allergen with PBMC collected from a subject and measuring the expression level of the GBP1 gene before and after the allergen contact.
  • a subject whose expression level of the GBP1 gene in the sample after the allergen contact is significantly reduced compared to the expression level before the allergen contact is suspected of having an allergic disease.
  • patients with allergic diseases compared to healthy subjects 1 are patients with allergic diseases compared to healthy subjects 1
  • the GBP1 gene that can be used as an indicator of an allergic disease is referred to as an indicator gene.
  • the indicator gene can be combined with not only GBP1 but also other genes that are indicators of allergic diseases. By combining and measuring a plurality of genes as indicator genes, detection accuracy can be improved. Since allergic '14 disease patients such as bronchial asthma patients are a heterogeneous population, a more reliable diagnosis can be made by using multiple genes as indices.
  • the expression level of the indicator gene includes transcription of the gene into mRNA and translation into protein. Therefore, the method of testing for allergic diseases by the present invention is performed based on the comparison of the expression intensity of mRNA corresponding to the indicator gene or the expression level of the protein encoded by the indicator gene.
  • the average value of the change rate of the healthy subject is set based on the change rate of the expression level of the healthy subject at the time of allergen stimulation of the indicator gene.
  • the rate of change is the ratio of the expression level of the indicator gene before and after antigen stimulation.
  • a range of ⁇ 2 S.D. of the average value is set as a standard value.
  • the expression level of the indicator gene in the test for allergic disease in the present invention can be measured according to a known gene analysis method. Specifically, for example, a hybridization technique using a nucleic acid that hybridizes to the gene as a probe or a gene amplification technique using a DNA that hybridizes to the gene of the present invention as a primer can be used.
  • the probe or primer used in the test of the present invention can be set based on the base sequence of the indicator gene.
  • the nucleotide sequence of the human GBP1 gene is known as GenBank Acc. No. M55542.
  • the nucleotide sequence of the human GBP1 gene is shown in SEQ ID NO: 1, and the amino acid sequence encoded by the nucleotide sequence described in SEQ ID NO: 1 is shown in SEQ ID NO: 2.
  • genes in higher animals are frequently associated with polymorphisms. Also, there are many molecules that produce isoforms composed of mutually different amino acid sequences during the splicing process. Even if the gene has a mutation in the nucleotide sequence due to the polymorphic isoform, any gene that has the same activity as the indicator gene and is involved in allergy is included in the indicator gene of the present invention.
  • a polynucleotide comprising the nucleotide sequence of the indicator gene or a polynucleotide containing at least 15 nucleotides complementary to a complementary strand thereof can be used.
  • the “complementary strand” refers to one strand of a double-stranded DNA consisting of A: T (U for RNA) and G: C base pairs with respect to the other strand.
  • the term “complementary” is not limited to a case where the sequence is completely complementary to at least 15 contiguous nucleotide regions, and is at least 70%, preferably at least 80%, more preferably 90%, and still more preferably 95%. It is only necessary to have the above homology on the base sequence.
  • the homology of the nucleotide sequences can be determined by an algorithm such as BLAST.
  • Such a polynucleotide can be used as a probe for detecting the indicator gene and as a primer for amplifying the indicator gene.
  • a primer When used as a primer, it usually has a chain length of 15 bp to 100 bp, preferably 15 bp to 35 bp.
  • a DNA having at least a part or all of the sequence of the indicator gene (or its complementary strand) and having a chain length of at least 15 bp is used.
  • the 3′-side region needs to be complementary, but a restriction enzyme recognition sequence tag or the like can be added to the 5′-side.
  • the “polynucleotide” in the present invention can be DNA or RNA.
  • oligonucleotide means a polynucleotide having a relatively low degree of polymerization among polynucleotides. Oligonucleotides are included in polynucleotides.
  • Testing for allergic diseases using the hybridization technique can be performed using, for example, a Northern hybridization method, a dot plot method, a method using a DNA microarray, or the like.
  • gene amplification techniques such as the RT-PCR method can be used.
  • the RT-PCR method in the process of gene amplification: By using the PCR amplification monitoring method, it is possible to perform more quantitative analysis on the expression of the gene of the present invention.
  • PCR amplification monitoring method for example, ABI PRISM7700 (PE Biosystems) can be used.
  • the method for detecting an allergic disease of the present invention can also be carried out by detecting a protein encoded by the indicator gene. 'Hereinafter, in the present specification, the protein encoded by the indicator gene is referred to as an indicator protein.
  • a test method for example, a western plotting method, an immunoprecipitation method, an ELISA method, or the like using an antibody that binds to these indicator proteins can be used.
  • Antibodies that bind to the indicator protein used for this detection can be obtained using techniques well known to those skilled in the art.
  • the antibody used in the present invention can be a polyclonal antibody or a monoclonal antibody (Milstein C, et al., 1983, Nature 305: 934): 537-40).
  • a polyclonal antibody against an indicator protein is obtained by extracting blood of a mammal sensitized with an antigen and separating serum from the blood by a known method.
  • polyclonal antibody serum containing the polyclonal antibody can be used. Alternatively, if necessary, a fraction containing a polyclonal antibody can be further isolated from the serum. In addition, to obtain a monoclonal antibody, immunocytes are removed from a mammal sensitized with the above antigen, and are fused with myeloma cells and the like. The hybridoma obtained in this way is clawed and the antibody is isolated from the culture. Can be recovered as a monoclonal antibody.
  • these antibodies may be appropriately labeled and used. Further, without labeling the antibody, a substance that specifically binds to the antibody, for example, protein A or protein G can be labeled and detected indirectly. As a specific detection method, for example, an ELISA method can be mentioned.
  • a protein or a partial peptide thereof used as an antigen can be prepared by, for example, incorporating the gene or a part thereof into an expression vector, introducing this into an appropriate host cell, preparing a transformant, and culturing the transformant.
  • To express the recombinant protein and the expressed recombinant protein can be purified from a culture or a culture supernatant.
  • an oligonucleotide comprising an amino acid sequence encoded by these genes or a partial amino acid sequence of an amino acid sequence encoded by a full-length cDNA can be chemically synthesized and used as an immunogen.
  • allergic diseases can be tested using not only the expression level of the indicator gene but also the activity of the indicator protein in a biological sample as an indicator.
  • the activity of the indicator protein refers to the biological activity of each protein.
  • the activity of the indicator protein can be detected based on a known method.
  • a biological sample of a subject is usually used as a sample.
  • PBMC peripheral blood mononuclear cells
  • monocytes and the like can be used as a biological sample.
  • a monocyte cell refers to a cell obtained by dividing a monocyte. More specifically, monocyte cells include macrophage-dendritic cells that have been differentiated with the transition of monocytes to tissues. In addition, monocytic cells such as THP-1 cells that have been established while maintaining the characteristics of monocytes are also included.
  • PBMC can be collected by diluting peripheral blood and centrifuging using Ficoll.
  • the test method of the present invention can be carried out by measuring the indicator gene or indicator protein using the separated PBMC as a sample.
  • blood, sputum, nasal mucosal secretions, bronchoalveolar lavage fluid, lung scraper cells, and the like can also be used as the biological sample in the present invention. Methods for collecting these biological samples are also known.
  • PBMC or whole blood as a sample, blood cells are destroyed and the indicator gene mRNA or indicator protein in the cells is measured.
  • the expression level of an indicator gene is measured before and after stimulating PBMC with an allergen.
  • an allergic immune response actually occurs by measuring the amount of marker gene protein production or gene transcription in PBMC or monocytes, T cells, or neutrophils contained in PBMC. You can judge the strength and the strength. This makes it possible to confirm that the added antigen is an allergen and to estimate the severity of the disease.
  • allergenic substances can be used as the allergen that can be used in the present invention. Specifically, mites, house dust, plant pollen, or various food-derived proteins are known as allergenic substances. These allergens may be derived from nature or may be synthesized by a method such as genetic recombination. Allergens can also be fragments of proteins. Methods for preparing purified allergens are also known.
  • test method of the present invention can be performed based on the stimulus by the typical allergen as described above.
  • the test method of the present invention can be used to identify allergens to which a certain patient responds particularly high. Identifying substances in patients with allergic diseases that show an excessive immune response and isolating patients from these substances is an important issue in controlling allergic symptoms. That is, the present invention relates to a method for evaluating allergen activity comprising the following steps, wherein the indicator gene is GBP1.
  • PBMCs are stimulated by the compound acting as an allergen. It has been clarified that the expression level of the indicator gene decreases. Therefore, the degree of decrease in the expression level of the indicator gene in PBMC that has been contacted with a compound reflects the allergen activity of the compound. That is, the magnitude of the allergen activity is directly proportional to the degree of decrease in the expression level of the indicator gene.
  • the allergen activity of the compound to be evaluated for allergen activity is determined to be large. You. Conversely, if the degree of the decrease in the expression level is not significantly different from that of the healthy subject, it indicates that the compound does not have allergen activity against the PBMC.
  • the method for evaluating allergen activity of the present invention can be performed in vitro without exposing a patient to a test compound.
  • the method for evaluating allergen activity according to the present invention can be said to be a safe test with less invasiveness to the subject as compared with a known scratch test or the like.
  • the expression level of the indicator gene can be easily measured by hybridization—PCR or Imnoassay.
  • the present invention can be said to be a test that can be easily performed as compared with a known lymphocyte blastogenesis test or the like.
  • the rate of change of the indicator gene in each compound is determined based on the evaluation method of the present invention.
  • the rate of change is compared between the compounds, and a compound having a large rate of change is considered to be a compound that is likely to induce an immune response in the subject.
  • one particular compound can be used as the test compound to determine if the patient has an allergy to that compound. That is, the present invention is useful as a method for screening a compound having an allergic disease with a compound that acts as an allergen.
  • kits for evaluating allergen activity, comprising the following elements, wherein the indicator gene is GBP1.
  • a reagent for measuring the expression level of the indicator gene for example, a primer probe for detecting the mRNA of the indicator gene can be used. Further, an antibody capable of detecting a protein encoded by the indicator gene (indicator protein) can also be used.
  • any antigenic substance that may cause an allergic disease as described above can be used. Only one potential allergen can be combined with the kit. Alternatively, a plurality of compounds can be combined. It is convenient to add a test compound to a container for incubating PBMC in advance, since the evaluation method of the present invention can be carried out only by dispensing PBMC.
  • kits and culture media necessary for PBMC collection and culture can be further combined.
  • a compound having no possibility of acting as an allergen can be combined as a negative control, or a compound having a high possibility of acting as an allergen can be attached as a positive control.
  • stimulation can be performed by adding an allergen to isolated PBMC or the like. Allergen stimulation is transmitted to T cells that recognize this antigen through monocyte phagocytosis and antigen presentation, and an immune response is initiated.
  • the measured value of the expression level of the indicator gene in blood cells can be corrected by a known method. With the correction, changes in the expression level of the gene in the cells can be compared. Correction of the measured value is based on the measured value of the expression level of a gene (housekeeping gene) which is expressed in blood cells and whose expression level does not largely fluctuate irrespective of the state of the cell. It is performed by correcting the measured value of the expression level.
  • the indicator gene of the present invention showed a decrease in the expression level of PBMC collected from a patient having an allergic disease in vitro when the PBMC was stimulated with allergen in vitro. Therefore, an allergic disease such as bronchial asthma and diabetic dermatitis can be tested using the expression level of the indicator gene as an indicator.
  • the test for allergic disease in the present invention includes, for example, the following tests. Even if a patient cannot show an allergic disease by a general test while showing symptoms of bronchial asthma, the test based on the present invention can easily determine that he is a patient with an allergic disease. More specifically, a decrease in the expression of an indicator gene in a patient exhibiting a symptom suspected of having an allergic disease is attributed to the cause of the symptom. This indicates that the disease is highly likely to be a lugi monogenic disease. Some bronchial asthma is caused by an allergic reaction, while others are not. Diagnosis of the causes of bronchial asthma is a very important therapeutic step because the treatment methods for patients are quite different. The detection method of the present invention can provide extremely important information in identifying the cause of bronchial asthma.
  • tests can be done to determine if allergic symptoms are improving.
  • the expression level of the indicator gene of the present invention was reduced in cells contained in the PBMC when PBMC collected from a patient having an allergic disease in vitro was stimulated with allergen.
  • T cells are cells that act as control towers in the immune response. Monocytes also play an important role in presenting antigens to T cells. Therefore, in PBMC stimulated by an allergen, a gene whose expression fluctuates in T cells responsible for its immune response is useful for determining the therapeutic effect. More specifically, a decrease in the expression of an indicator gene in a patient diagnosed with an allergic disease indicates that the allergic symptoms are likely to be progressing.
  • the present invention also relates to an animal model for an allergic disease comprising a transgenic non-human animal in which the expression level of an indicator gene in T cells or monocytes is reduced.
  • Animal models of allergic diseases are useful for elucidating in vivo changes in bronchial asthma.
  • the allergic disease model animal of the present invention is useful for evaluating a therapeutic agent for allergic bronchial asthma.
  • the expression level of the indicator gene is reduced in cells contained in the PBMC.
  • an animal in which the expression level of the GBP1 gene or a gene functionally equivalent to the GBP1 gene in these cells is artificially suppressed can be used as a model animal for an allergic disease.
  • the decrease in the expression level in T cells and monocytes is defined as Includes reduced levels of target gene expression throughout the vesicle. That is, the expression level of the gene is decreased not only by T cells and Z cells or monocytes, but also in whole blood cells or systemically. Including cases.
  • a functionally equivalent gene is a gene that encodes a protein having an activity similar to that of the protein encoded by the indicator gene.
  • a representative example of a functionally equivalent gene is a counterpart of an indicator gene in a transgenic animal that the transgenic animal originally has. For example, rats (Deborah J et al, Rat p67 GBP is induced by interferon-gamma and isoprenoid-modified in macrophages;
  • GBP1 of geranylgeranyl isoprenoid modification of an interferon-gamma-inducible guanosine triphosphate-binding protein is known.
  • a gene encoding a protein having a homology of, for example, 90% or more, preferably 95% or more, more preferably 99% or more with the amino acid sequence of the human GBP1 protein is a gene that is functional with the GBP1 gene.
  • a gene that can be amplified using the oligonucleotide having the nucleotide sequence of SEQ ID NO: 3 or SEQ ID NO: 4 used as a primer in a cell contained in PBMC upon stimulation with an allergen Genes whose expression is reduced are also functionally equivalent genes.
  • a gene that hybridizes under stringent conditions with a gene encoding human GBP1 is a functionally equivalent gene in the present invention.
  • the following conditions can be generally shown as stringent conditions. That is, 4xSSC! , 65. C. Hybridize with O.lxSSC And wash at 65 ° C for 1 hour.
  • the temperature conditions for the hybridization and washing which greatly affect the stringency, can be adjusted according to the melting temperature (Tm). Tm varies depending on the ratio of constituent bases to the base pairs to be hybridized, and the composition of the hybridization solution (salt concentration, sodium formamide-dodecyl sulfate concentration). Therefore, those skilled in the art can experimentally or empirically set conditions that give the same stringency in consideration of these conditions.
  • PBMCs collected from patients with allergic diseases in vitro are stimulated with allergen
  • a gene whose expression is reduced in cells contained in the PBMCs suppresses the allergic immune response Genes that make up the transmission system for communication.
  • the result of the loss of control of the immune response to allergen stimulation due to the decreased expression of these genes is manifested as an allergic symptom.
  • the genes whose expression is reduced in cells contained in the PBMCs are responsible for the allergic immune response in PBMCs. It is a gene that plays an important role in regulation. Therefore, drugs that enhance the expression or enhance the activity of this gene, in the treatment of allergies, not only improve the allergic symptoms but also eliminate the essential cause of allergic pathogenesis. Can be expected.
  • the genes whose expression is reduced in cells contained in PBMCs when stimulated with allergens from PBMCs collected from patients with allergic diseases in vitro have important significance. Therefore, it is of great significance to use transgenic animals, which can be obtained by reducing the expression level of this gene, as animal models for allergic diseases to evaluate the role of the gene and drugs that target the gene. There is.
  • the animal model for an allergic disease can be used in addition to a drug for treating or preventing an allergic disease, which will be described later. It is useful for elucidating the mechanism of the disease and for testing the safety of the screened compounds.
  • the animal model animal for allergic disease develops bronchial asthma or shows a change in a measurement value associated with any allergic disease
  • a screening system for searching for a compound having an action to restore it can be constructed.
  • a decrease in the expression level means either a state in which transcription of an indicator gene provided in the host and translation into a protein are inhibited, or a state in which degradation of a protein as a translation product is promoted. I do.
  • the expression level of the gene can be confirmed, for example, by quantitative PCR as described in Examples.
  • the activity of the protein, which is a translation product, can be confirmed by comparing with the normal state.
  • transgenic animals can include animals in which the indicator gene has been knocked out, animals in which the gene has been replaced (knocked in), and the like.
  • transgenic animals into which antisense DNA against the indicator gene, DNA encoding lipozyme, DNA functioning as a decoy nucleic acid, or the like can be used as the transgenic animal in the present invention.
  • Other examples include animals in which a mutation has been introduced into the coding region of the indicator gene to suppress its activity or have been modified to an amino acid sequence that is easily degraded. Amino acid sequence mutations can indicate substitutions, deletions, insertions, or additions.
  • the expression itself of the indicator gene of the present invention can be regulated by mutating the transcriptional regulatory region of the gene.
  • transgenic animals for specific genes are known. That is, a method in which a gene and an egg are mixed and treated with calcium phosphate, or a method in which a gene is directly introduced into a nucleus of a pronuclear phase egg with a micropipette under a phase contrast microscope (microinjection method, US Pat.
  • Transgenic animals can be obtained by methods using embryonic stem cells (ES cells). Other methods include introducing a gene into a retrovirus vector and infecting the egg, and using sperm A sperm vector method for introducing a gene into an egg has also been developed.
  • the sperm vector method is a gene recombination method in which a spontaneous gene is attached to a sperm or incorporated into sperm cells by a method such as electroporation and then fertilized by an egg to introduce the foreign gene. (M. Lavitranoet et al. Cell, 57, 717, 1989).
  • the transgenic animal used as the allergic disease model animal of the present invention can be prepared using any vertebrate other than human. Specifically, transgenic animals in which various genes have been introduced or their expression levels have been modified in vertebrates such as mice, rats, rabbits, rabbits, miniature pigs, goats, sheep, and birds have been created. I have.
  • the present invention relates to a method for screening a therapeutic drug for an allergic disease.
  • a therapeutic drug for an allergic disease when the PBMCs collected from patients with allergic diseases are stimulated with allergens in vitro, the expression levels of the indicator genes are significantly reduced in cells contained in the PBMCs. Therefore, by selecting a compound that increases the expression level of these genes, a therapeutic agent for allergic diseases can be obtained.
  • a compound that increases the expression level of a gene is a compound that has an action that promotes any of the steps of gene transcription, translation, and protein activity expression.
  • the indicator gene of the present invention is a gene whose expression level is decreased in PBMC stimulated with allergen.
  • treatment is performed when the expression level of the indicator gene is reduced, that is, when allergic symptoms occur. Therefore, compounds that increase the expression of an indicator gene independently of allergen stimulation are useful as therapeutics for allergic diseases.
  • the screening method of the therapeutic agent for allergic diseases of the present invention can be performed in vivo or in vitro. This screening can be performed, for example, according to the following steps.
  • the indicator gene in the screening method of the present invention includes, in addition to the GBP1 gene, any gene functionally equivalent to the GBP1 gene. Including the gene.
  • test animal in the screening method of the present invention for example, an experimental animal having an indicator gene or an allergic disease model animal can be used as a test animal.
  • a drug candidate compound By administering a drug candidate compound to a test animal and monitoring the effect of the compound on the expression of the indicator gene in a biological sample of the test animal, the effect of the drug candidate compound on the expression level of the indicator gene can be detected. Fluctuations in the expression level of the indicator gene in the biological sample of the test animal can be monitored by the same method as the detection method of the present invention. Further, if a drug candidate compound that increases the expression level of the indicator gene is selected based on the result of this detection, the drug candidate compound can be screened.
  • the screening according to the present invention can be performed by collecting a biological sample from a test animal and comparing the expression level of the indicator gene with a control.
  • a biological sample whole blood, PBMC, T cells, monocytes, macrophages, dendritic cells, or the like can be used. Methods for collecting and preparing these biological samples are known.
  • drugs that participate in the expression of the indicator gene in various forms can be selected.
  • drug candidate compounds having the following actions can be found.
  • the screening according to the present invention can be performed by administering a candidate conjugate to this system and tracking the change in the expression level of the indicator gene of the present invention.
  • the present invention relates to the screening method, comprising a step of stimulating a test animal with an allergen before and / or after administration of the candidate compound.
  • the candidate compound is stimulated with an allergen before administration, the effect of the candidate compound on suppressing an immune response after the allergen stimulation can be detected.
  • Compounds obtained by such screening can be expected to have therapeutic effects on allergic diseases.
  • allergen is stimulated after administration of the candidate compound, the action of the candidate compound to suppress the initiation of an immune response by allergen stimulation can be detected.
  • Compounds obtained by such screening can be expected to have an effect of preventing allergic diseases.
  • Allergens that can be used in the screening method of the present invention include the allergens described above. When comparing the expression levels before and after allergen stimulation, a compound that does not show a significant decrease in the expression level after allergen stimulation can also be selected as a compound that increases the expression of the indicator gene.
  • a method of bringing a candidate compound into contact with cells expressing the indicator gene and selecting a compound that increases the expression level of the indicator gene can be used.
  • This screening can be performed, for example, according to the following steps.
  • cells that express the indicator gene can be obtained by inserting the indicator gene into an appropriate expression vector and introducing the vector into an appropriate host cell.
  • Usable vectors and host cells may be any as long as they can express the gene of the present invention. Examples of host cells in a host vector system include Escherichia coli, yeast, insect cells, animal cells, and the like, and vectors that can be used for each can be appropriately selected.
  • Examples of a method for introducing a vector into a host include a biological method, a physical method, and a chemical method.
  • Biological methods include, for example, a method using a viral vector, a method using a specific receptor, a cell fusion method (HVJ (Sendai virus), polyethylene daricol (PEG), an electric cell fusion method, a micronucleus, etc. Fusion method (transfer of chromosomes)).
  • Examples of the physical method include a microinjection method, an electoral opening method, and a method using a gene part take / regan (gene gun).
  • Chemical methods include the calcium phosphate precipitation method, the liposome method, the DEAE dextran method, the protoplast method, the erythrocyte ghost method, the erythrocyte membrane ghost method, and the microphone-mouth capsule method.
  • the cell expressing the indicator gene for example, the monocytic leukemia cell line THP-1 or the human acute leukemia T cell Jurkat (ATCC Number TIB-152) is suitable for the screening method of the present invention. It is known that THP-1 produces GBP1
  • a step of contacting with the allergen may be added before or after contacting the candidate compound with the cell.
  • a significant decrease in the indicator gene is not observed, or a compound that increases the gene can be selected.
  • the expression levels of the indicator genes can be compared not only with the expression levels of the proteins encoded by these genes, but also by detecting the corresponding mRNA.
  • the mRNA sample preparation step described above is performed instead of the protein sample preparation step. Detection of mRNA or protein can be performed by a known method as described above.
  • a transcriptional regulatory region of the indicator gene of the present invention can be obtained, and a reporter atssay system can be constructed.
  • the reporter accession system refers to an accession system that screens a transcriptional regulatory factor acting on the transcriptional regulatory region, using the expression level of a reporter gene located downstream of the transcriptional regulatory region as an index.
  • transcription control region examples include a promoter, an enhancer, and a CAAT pox, a TATA box, and the like which are usually found in a promoter region.
  • CAT chloramphenicol acetyltransferase
  • luciferase gene growth hormone gene, and the like can be used as islet genes.
  • a transcription regulatory region used for the screening of the present invention can be obtained as follows. That is, first, based on the nucleotide sequence of the indicator gene disclosed in the present invention (GenBank Acc.No.M55542), a method using PCR or hybridization is used from a human genomic DNA library such as a BAG library or a YAC library. Screening is performed to obtain a genomic DNA clone containing the sequence of the cDNA. Based on the sequence of the obtained genomic DNA, the transcription regulatory region of the cDNA disclosed in the present invention is estimated, and the transcription regulatory region is obtained. The obtained transcription regulatory region is The reporter construct is constructed by closing the clone so that it is located upstream of the target gene. The resulting reporter construct is introduced into a cultured cell line to obtain a transformant for screen Jung. By contacting a candidate compound with the transformant, a compound that controls the expression of a reporter gene can be screened.
  • Reporter assays utilizing the transcriptional regulatory region of GBP1 are known (Lew JD et al. OverlapOing elements in the guanylate-binding protein gene promoter mediate transcriptional induction by alpha and gamma interferons;
  • the transcriptional control region located upstream of the GBPl gene was ligated to the clonal ramphenicol acetinoletransferase (CAT) gene to create a reporter Atsuy system. According to this assay system, it was found that the promoter region contained ISRE (IFN-stimulated response element).
  • ISRE IFN-stimulated response element
  • the present invention relates to a method for screening a therapeutic agent for an allergic disease, comprising the following steps, wherein the indicator gene is GBP1 or a gene functionally equivalent to the GBP1 gene.
  • Step of selecting a compound that increases the activity of the protein as compared to a control GBP1 as an indicator protein in the present invention may be, for example, a GTPase (hereinafter abbreviated as GTPase) activity (Guenzi E. et al. al. The helical domain of GBP-1 mediates the inhibition of endothelial cell proliferation by inflammatory cytokines; EMBO Journal (2001) 20, 5568-5577) ⁇ Antiviral activity (Anderson SL et al.
  • GTPase GTPase activity
  • Interferon-induced guanylate binding protein-1 ( GBP-1) is known to have mediates an antiviral effect against vesicular stomatitis virus and encephalomyocarditis virus; Virology (1999) 256, 8-14).
  • GTPase activity can be measured by incubating the substrate GTP with the enzyme and separating and quantifying the resulting GDP or GMP by thin-layer chromatography.
  • a human-derived cell line is infected with vesicular stomatitis virus or encephalomyocarditis, virus particles are produced, and the number can be measured by a known method.
  • Antiviral activity is measured by the extent to which it reduces the number of virus particles produced.
  • kits Polynucleotides, antibodies, cell lines, or model animals required for various screening methods according to the present invention can be combined in advance to form a kit. More specifically, it is composed of, for example, cells that express the indicator genes and reagents for measuring the expression levels of these indicator genes.
  • a reagent for measuring the expression level of an indicator gene for example, a polynucleotide containing a nucleotide sequence of at least one indicator gene or at least 15 nucleotides having a nucleotide sequence complementary to a complementary strand thereof is used. Oligonucleotides are used. Alternatively, an antibody that recognizes a peptide containing an amino acid sequence of at least one indicator protein can be used as a reagent.
  • These kits are packaged with the substrate compound used to detect the label, media and containers for cell culture, positive and negative standard samples, and instructions describing how to use the kit. You can also put it.
  • Test candidate compounds used in these screenings include steroid derivatives and other compound samples synthesized by existing chemical methods, compound compounds synthesized by combinatorial chemistry, extracts of animal and plant tissues, or microbial cultures. And a mixture containing a plurality of compounds such as a product, and a sample purified therefrom.
  • the expression level of the indicator gene is significantly reduced in cells contained in PBMCs obtained by stimulating in vitro PBMCs collected from patients with allergic diseases with allergens. Therefore, a drug that promotes the expression or enhances the activity of this gene will not only ameliorate allergic symptoms but also eliminate the essential cause of allergic pathogenesis in the treatment of allergy. Can be expected.
  • the therapeutic effect of one allergic symptom can be achieved by supplementary administration of the protein encoded by these indicator genes.
  • the protein can be supplemented by administering the protein itself or by introducing a vector capable of expressing the indicator gene into a patient by a gene therapy technique.
  • the present invention relates to a therapeutic agent for an allergic disease, comprising, as an active ingredient, an indicator gene or a protein encoded by the indicator gene, wherein the indicator gene is a GBP1 gene or a gene functionally equivalent to GBP1.
  • a therapeutic drug comprising, as an active ingredient, an indicator gene or a protein encoded by the indicator gene, wherein the indicator gene is a GBP1 gene or a gene functionally equivalent to GBP1.
  • the compound selected by the screening method of the present invention, the indicator gene, or the protein encoded by the indicator gene is useful as a therapeutic drug for allergic diseases.
  • the remedy for allergic diseases of the present invention can be produced by containing these components as active ingredients and mixing them with a physiologically acceptable carrier, excipient, diluent or the like.
  • the therapeutic agent for an allergic disease of the present invention can be administered orally or parenterally for the purpose of improving allergic symptoms.
  • Oral preparations include granules, powders, tablets, capsules, solvents,?
  • a dosage form such as a suspending agent or a suspending agent can be selected.
  • Injections include subcutaneous injections, intramuscular injections, and intraperitoneal injections.
  • a therapeutic effect can be achieved by introducing a gene encoding the protein into a living body using a gene therapy technique. Introducing genes encoding proteins that produce therapeutic effects to living organisms Techniques for treating diseases by insertion and expression are known.
  • the GBP1 gene can be incorporated downstream of an appropriate promoter sequence and administered as a GBP1 expression vector.
  • this expression vector is introduced into T cells or monocytes of a patient with an allergic disease, the GBP1 gene is expressed in these cells, and an allergic therapeutic effect can be achieved by increasing the expression level of the gene.
  • a method for introducing an expression vector into a cell a method using iu viVo or ex Wvo is known.
  • the dosage varies depending on the age, sex, weight and condition of the patient, therapeutic effect, administration method, processing time, or the type of active ingredient contained in the pharmaceutical composition, but is usually once per adult.
  • FIG. 1 shows the results of comparing the expression levels of GBP1 in PBMC before and after mite antigen stimulation.
  • the vertical axis shows the ratio of the copy number of GBP1 mRNA per 1 ng of PBMC RNA, log2 (Z stimulation without mite stimulation) — and the horizontal axis shows the mite antigen-specific IgE score.
  • the upper graph shows the results of PBMCs derived from patients, and the lower graph shows the results of PBMCs derived from healthy subjects.
  • FIG. 2 is a view showing the results of comparing the expression levels of GBP1 in various peripheral blood leukocytes.
  • the vertical axis shows the copy number of GBP1 mRNA per 1 ng of RNA, and the horizontal axis shows the type of white blood cells.
  • IgE Specific IgE was measured by the CAP RAST method, which was a modification of the RAST method using a paper disk as a solid phase.
  • the IgE antibody titer of each sample was determined based on a serum containing a standard antibody titer manufactured by Pharmacia. The obtained values were indicated by scores.
  • Table 2 shows the scores of the mite-specific IgE antibody titers of the subjects. As shown in Table 2, more than half of the healthy subjects scored 2 or less. On the other hand, a high score was seen in the patient group, indicating that they had allergies to the mite antigen.
  • PBMC peripheral blood mononuclear cell
  • PBMC peripheral blood mononuclear cell
  • the needle was removed and transferred to a 50 ml centrifuge tube (made of polypropylene). After centrifugation at 1500 rpm for 5 minutes at room temperature, 1.1 ml was collected from the surface as close as possible, and centrifuged at 15000 rpm for 5 minutes at 4 ° C to collect 1 ml of supernatant as plasma.
  • Du antigen is a typical allergen.
  • To find an allergic disease-related gene that responds to mite antigen stimulation we stimulated PBMCs from allergic disease patients and healthy subjects with mite antigen proteins, respectively, and searched for genes that show different expression fluctuations in both.
  • PBMCs were prepared from 100 mL of peripheral blood of one patient with an allergic disease and one healthy subject according to the method described in Example 2. The obtained PBMC is used for the mite extract
  • the cells were cultured under three conditions: in the presence of the mite extract and dexamethasone (100 nM), or in the absence of the control.
  • RPMI1640 medium containing 10% fetal calf serum, penicillin (100 units / ml), and streptomycin (100 / ig / ml) was used.
  • PBMCs were collected, and T cells (CD3-positive cells) and monocytes (CD14-positive cells) were separated.
  • T cells and monocytes were sequentially labeled with anti-CD3 antibody magnetic beads and anti-CD14 antibody magnetic beads, and separated using a magnetic cell separator (Milteni, Biotec). RNA was extracted from the obtained cells, and gene expression analysis was performed using an oligonucleotide array GeneChip HuGene FL Array (Affymetrix).
  • M55542 was pitched on the oligonucleotide array as Human guanylate binding protein isoform I.
  • DU antigen was stimulated using PBMC prepared from the peripheral blood of the subjects shown in Table 2, and the expression level of GBP1 was measured by quantitative RT-PCR. did. Stimulation was performed in the same manner as above, RNA was prepared from the whole collected PBMC, and the culture supernatant was preserved. After cDNA was prepared from RNA by a conventional method, quantitative RT-PCR was performed using # 7700 (Applied Biosystems) using a primer and probe specific for GBP1. Primers and probes were designed based on the GBP1 sequence registered on the chip.
  • M55542-f TGGCATCAGAGATCCACATGA (SEQ ID NO: 3)
  • ⁇ 55542- ⁇ GGCTGTGTAATGGCAGAAAGG (SEQ ID NO: 4)
  • M55542p AGGCCCAATGTGCCTCATTGAGAACAC (SEQ ID NO: 5)
  • the probe for quantitative determination of GBP1 has FAM (6-carboxy-fluorescein) at the 5 ′ end and TAMRA (6-carboxy-N, N, N ′, N ′) at the 3 ′ end. -TetramethyMiodamine).
  • a plasmid in which a 109 bp sequence to be amplified with the primer was cloned was used as a copy number standard.
  • Table 1 shows the composition of the reaction mixture for monitoring PCR amplification.
  • Table 2 shows the quantitative results.
  • FIG. 1 shows the results of comparing the expression levels of the GBP1 gene of the same PBMC before and after mite antigen stimulation.
  • the ratio of the RNA copy number in the PBMC after stimulation to the RNA copy number in the PBMC before stimulation is logarithmically displayed. Therefore, if the expression level decreases after stimulation, a negative value is displayed.
  • Reaction composition of ABI-PRISM 7700 (reaction volume per 1 ⁇ ) (Sterile distilled water 23.75 L)
  • the expression level of GBP1 tended to decrease in the group of patients with allergic diseases when stimulated with mite antigen compared to the group of healthy subjects (FIG. 1).
  • the expression changes caused by mite antigen stimulation significant changes in expression were observed between healthy subjects with a mite-specific IgE score of 0 and patient samples with a specific mite IgE score of 5 (p ⁇ 0.01).
  • T cells B cells, monocytes, neutrophils, and eosinophils prepared from peripheral blood of five healthy subjects was quantified.
  • a 3% dextran solution was added to whole blood collected from the subject and left at room temperature for 30 minutes to sediment erythrocytes.
  • the leukocyte fraction in the upper layer was collected, placed on a ficoll solution (Ficoll-Paque PLUS; Amersham Pharmacia Biotech), and centrifuged at 1500 rpm for 30 minutes at room temperature.
  • the granulocyte fraction collected in the lower layer was reacted with CD16 antibody magnetic beads at 4 ° C for 30 minutes, and cells eluted without being trapped by separation using MACS were used as eosinophils in the experiment.
  • Neutrophils were prepared by eluting eosinophils and then eluting and recovering cells trapped by CD16 antibody magnetic beads out of the magnetic field.
  • the mononuclear cell fraction collected in the intermediate layer by Ficoll centrifugation is eluted by MACS CD3 antibody magnetic beads and the fraction trapped (mixture of monocytes and B cells) (T cell fraction) ).
  • the eluted fraction was separated by MACS CD14 antibody magnetic beads into an eluted fraction (B cell fraction) and a trapped fraction (monocyte fraction), each of which was purified T cells, purified B cells, Then, purified monocytes were obtained.
  • -37-Eosinophils are solubilized using Isogen, neutrophils, T cells, B cells, and monocytes using RNeasy (Qiagen), after total RNA extraction and DNase treatment (the method is as described above). It was subjected to expression analysis. The primers, probes, etc. used are the same as above. Figure 2 shows the measurement results. As a result, high expression of GBP1 was observed particularly in monocytes, T cells, and neutrophils.
  • a gene GBP1 whose expression level in T cells contained in PBMC is significantly reduced when PBMC collected from a patient having an allergic disease in Wtro is stimulated with an allergen. Based on the indicator gene of the present invention, it has become possible to detect allergic diseases and screen therapeutic drugs.
  • the expression level of the allergic disease-related gene provided by the present invention can be easily known regardless of the type of allergen. Therefore, the pathology of allergic reaction can be comprehensively grasped.
  • the method of testing for allergy according to the present invention can analyze the expression level of peripheral blood leukocytes as a sample, and therefore has low invasiveness to patients. Moreover, for gene expression analysis, it is possible to perform highly sensitive measurement using a small amount of sample, unlike protein measurement such as ECP. Genetic analysis technology has been increasing its throughput and lowering its price year by year. Therefore, the method of testing for allergy according to the present invention is expected to be an important diagnostic method at the bedside in the near future. In this sense, the diagnostic value of these disease-related genes is high.
  • the present invention has provided a method for evaluating the allergen activity of a test substance on a subject.
  • the method of the present invention can be carried out in vitro using PBMC collected from a subject. That is, according to the present invention, the effect of the test substance on the subject can be easily evaluated without exposing the subject to the allergen.
  • the screening method of the present invention is directed to a method for stimulating a gene whose expression level in T cells contained in PBMC is significantly reduced when PBMC collected from a patient having an allergic disease in vitro is stimulated with an allergen. It is implemented as an indicator. T cells are cells that control the allergic immune response to allergens. Therefore, compounds that can be found by this screening method can be expected to be useful for controlling a wide variety of allergic conditions.

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Abstract

GBP1 est un gène identifié qui présente un taux d'expression sensiblement réduit dans des cellules contenues dans les monocytes du sang périphérique d'un patient atteint d'une maladie allergique, lorsque les PBMC sont stimulés à l'aide d'un allergène d'acarien in vitro. Ce gène peut être utile dans l'examen d'une maladie allergique et dans le criblage de médicaments pour traiter celle-ci ; il peut aussi servir d'indicateur pour évaluer une activité allergène, et facilite le criblage de composés utiles dans le traitement de la maladie allergique.
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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999010536A1 (fr) * 1997-08-22 1999-03-04 Yale University Procede d'etude des modifications de l'expression des genes dans les granulocytes
WO2000012737A1 (fr) * 1998-08-26 2000-03-09 Bavarian Nordic Research Institute A/S Proteine 1 de fixation du guanylate (gpb-1) inhibiteur de la proliferation cellulaire et marqueur pour la determination du stade de differentiation cellulaire

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999010536A1 (fr) * 1997-08-22 1999-03-04 Yale University Procede d'etude des modifications de l'expression des genes dans les granulocytes
WO2000012737A1 (fr) * 1998-08-26 2000-03-09 Bavarian Nordic Research Institute A/S Proteine 1 de fixation du guanylate (gpb-1) inhibiteur de la proliferation cellulaire et marqueur pour la determination du stade de differentiation cellulaire

Non-Patent Citations (3)

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Title
GUENZI E. ET AL.: "The helical domain of GBP-1 mediates the inhibition of endothelial cell proliferation by inflammatory cytokines", EMBO J., vol. 20, no. 20, 15 October 2001 (2001-10-15), pages 5568 - 5577, XP002966002 *
PRAKASH B. ET AL.: "Triphosphate structure of guanylate-binding protein 1 and implications for nucleotide binding and GTPase mechanism", EMBO J., vol. 19, no. 17, 1 September 2000 (2000-09-01), pages 4555 - 4564, XP002966003 *
SCHWAMMLE M. ET AL.: "Chicken guanylate-binding protein. Conservation of the GTPase activity and induction by cytokines", J. BIOL. CHEM., vol. 271, no. 17, 26 April 1996 (1996-04-26), pages 10304 - 10308, XP002966004 *

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