Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2001-05-22
2004-07-13
Johannsen, Diana B. (Department: 1634)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091200, C435S091510, C435S189000, C435S194000, C435S196000, C536S023200, C536S023500, C536S024310, C530S350000
Reexamination Certificate
active
06762021
ABSTRACT:
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a diagnostic reagent and a method for diagnosing Crohn's disease.
BACKGROUND OF THE INVENTION
Autoimmune diseases refer to a phenomenon wherein a biological defense system (immune system) attacks the cells of its own. Antibodies and lymphocytes reactive with the autoantigen are derived, which in turn develops tissue disorders and lesions. The autoimmune diseases are generally divided into two groups: those without organ specificity and those with organ specificity. The mechanism of the onset of the autoimmune diseases is mostly unclear, though involvement of autoimmunity is suggested. There are many problems to be solved such as diagnosis method and the like, which include the mechanism of the onset of autoimmune diseases.
The autoimmune diseases include Crohn's disease, as one of the diseases whose etiology has not been elucidated, in which an immune reaction against autoantigen and allergy are considered to be involved. This disease is an inflammatory bowel disease associated with inflammatory changes throughout the full thickness of the wall of the digestive tract, discontinuous deep ulcer and histologically noncaseating granuloma. The skipping of the lesion also characterizes this disease. The definite diagnosis of Crohn's disease is based on a comprehensive observation of the disease state, X rays, endoscopy and tissue images. However, since the diagnosis is possible only after the progress of the disease, earlier diagnosis is desired. In addition, differential diagnosis from other inflammatory bowel diseases, such as acute or chronic appendicitis, tuberculosis of the intestine, ulcerative colitis, ischemic enteritis and the like, is required.
There are reports documenting that the genes of membrane proteins such as interleukin 2 (IL-2) receptor, transferrin receptor, E-selectin (also referred to as ELAM-1, endothelial leukocyte adhesion molecule-1), VCAM-1 (vascular cell adhesion molecule-1), L-selectin, CD11, OX40, OX40 ligand and the like, and cytokines and chemokines such as interleukin 1&bgr; (IL-1&bgr;), IL-2, interleukin 6 (IL-6), interleukin 15 (IL-15), tumor necrosis factor a (TNF-&agr;), interleukin 18 (IL-18), interleukin 8 (IL-8), MCP-1, ENA-78 and the like are up-regulated in Crohn's disease, from a molecular biological approach taking note of the expression of cytokines and adhesion molecules. However, most of these genes in the reports showed the up-regulation due to a non-specific immune response associated with the inflammation observed in Crohn's disease, and the up-regulation is not specific to Crohn's disease.
SUMMARY OF THE INVENTION
It is therefore an object of the present invention to provide a method useful for diagnosing Crohn's disease and a reagent for the diagnosis.
In view of the above-mentioned problems, the present inventors have conducted intensive studies of the expression profiles at the gene level of Crohn's disease, in an attempt to enable early diagnosis of Crohn's disease and differential diagnosis from other diseases. Since a lesion and a non-lesion part can be clearly distinguished visually in Crohn's disease, a differential display method (Liang, P., and Pardee, A. B. Science 257:967-971 (1992), Liang, P., and Pardee, A. B. Curr. Opin. Immunol. 7:274-280 (1995)), wherein the genes expressed in the lesion and the non-lesion part in the same individual can be compared, was employed to compare gene expression profiles in the lesion and the non-lesion part. As a result, the expression of a certain kind of gene in the lesion was found to have been specifically potentiated and the gene could be identified, which resulted in the completion of the present invention.
Accordingly, the present invention provides the following.
(1) A reagent for diagnosing Crohn's disease, which contains at least one member selected from the group consisting of (i) a substance having a specific affinity for a gene of a type 6 protein phosphatase regulated by interleukin 2 (type 6 protein phosphatase regulated by IL-2; hereinafter to be also referred to as PP6 regulated by IL-2), (ii) a substance having a specific affinity for a gene of a Traf 2 and Nck interacting kinase (hereinafter to be also referred to as TNIK), (iii) a substance having a specific affinity for a gene of a FLICE inhibitory protein (hereinafter to be also referred to as FLIP), and (iv) a substance having a specific affinity for a gene of a glucocorticoid receptor &agr; (hereinafter to be also referred to as GR&agr;).
(2) The reagent for diagnosing Crohn's disease according to the above-mentioned mentioned (1), which further contains at least one member selected from the group consisting of (v) a substance having a specific affinity for a cytochrome oxidase subunit I gene and (vi) a substance having a specific affinity for a cytochrome b gene.
(3) The reagent for diagnosing Crohn's disease according to the above-mentioned (1) or (2), wherein the substance having a specific affinity is an oligonucleotide or polynucleotide probe, or an oligonucleotide or polynucleotide primer pair.
(4) A reagent for diagnosing Crohn's disease, which contains at least one member selected from the group consisting of (i) a substance having a specific affinity for PP6 regulated by IL-2, (ii) a substance having a specific affinity for TNIK, (iii) a substance having a specific affinity for FLIP, and (iv) a substance having a specific affinity for GR&agr;.
(5) The reagent for diagnosing Crohn's disease according to the above-mentioned (4), which further contains at least one member selected from the group consisting of (v) a substance having a specific affinity for a cytochrome oxidase subunit I and (vi) a substance having a specific affinity for cytochrome b.
(6) The reagent for diagnosing Crohn's disease according to the above-mentioned (4) or (5), wherein the substance having a specific affinity is an antibody or a fragment thereof.
(7) A method for diagnosing Crohn's disease, which comprises the steps of
(a) taking a biological sample from an animal that developed or is associated with a risk of developing Crohn's disease, and
(b) analyzing the expression of at least one gene selected from the group consisting of a gene of PP6 regulated by IL-2, a TNIK gene, a FLIP gene and a GR&agr; gene, in a biological sample thereof.
(8) The method for diagnosing Crohn's disease according to the above-mentioned (7), which further includes analyzing the expression of at least one gene selected from the group consisting of a cytochrome oxidase subunit I gene and a cytochrome b gene.
(9) A method for diagnosing Crohn's disease, which comprises the steps of
(a) taking a biological sample from an animal that developed or is associated with a risk of developing Crohn's disease, and
(b) analyzing the expression of at least one protein selected from the group consisting of PP6 regulated by IL-2, TNIK, FLIP and GR&agr;, in a biological sample thereof.
(10) The method for diagnosing Crohn's disease according to the above-mentioned (9), which further includes analyzing the expression of at least one protein selected from the group consisting of cytochrome oxidase subunit I and cytochrome b.
(11) The method for diagnosing Crohn's disease according to any of the above-mentioned (7) to (10), wherein the biological sample is an ileum tissue or colon tissue derived from the animal.
DETAILED DESCRIPTION OF THE INVENTION
The gene in the present invention may be of any form unless otherwise particularly specified. For example, complementary DNA (cDNA) prepared from mRNA and the like are included besides mRNA.
The respective elements that may be contained in the diagnostic reagent according to the present invention are explained in detail in the following.
(i) PP6 Regulated by IL-2 [Type 6 Protein Phosphatase Regulated by IL-2; Protein (36 kDa) described in Filali, M., et al., J. Cell. Biochem. 73:153-163 (1999)]
The PP6 regulated by IL-2 is a phosphoprotei
Tokunaga Katsushi
Tsuchiya Naoyuki
Johannsen Diana B.
Mitsubishi Pharma Corporation
Wenderoth , Lind & Ponack, L.L.P.
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