| PATENT ASSIGNEE'S COUNTRY | Japan |
| PATENT NUMBER | This data is not available for free |
| PATENT GRANT DATE | 09.05.2000 |
| PATENT TITLE |
Neutrophil chemotactic lymphokine, and method for the diagnosis of drug hypersensitive granulocytopenia using the same |
| PATENT ABSTRACT | The invention provides a neutrophil chemotactic lymphokine having an isoelectric point at 6.8-7.0, a cell line producing the lymphokine, and an agent for diagnosing drug-induced granulocytopenia, which comprises the lymphokine. When the diagnostic agent according to the invention is used for a patient, whether the patient has a possibility that drug-induced granulocytopenia may be caused or not can be diagnosed prior to the administration of a drug. Therefore, the patient can be safely treated without causing any side effect |
| PATENT INVENTORS | Hirashima; Mitsuomi (Takamatsu, JP) |
| PATENT ASSIGNEE | This data is not available for free |
| PATENT FILE DATE | March 25, 1998 |
| PATENT CT FILE DATE | September 26, 1996 |
| PATENT CT NUMBER | This data is not available for free |
| PATENT CT PUB NUMBER | This data is not available for free |
| PATENT CT PUB DATE | 03.04.97 |
| PATENT FOREIGN APPLICATION PRIORITY DATA | This data is not available for free |
| PATENT REFERENCES CITED |
Japanese Journal of Allergology, "Allergy", vol. 44, No. 8,Aug. 1995 (with English translation). The Abstract #665, Volume for the Ninth International Congress of Immunology, Jul. 1995, San Francisco, CA Nishi Yama et al. |
| PATENT CLAIMS |
What is claimed is: 1. An isolated neutrophil chemotactic lymphokine having an isoelectric point at 6.8-7.0, wherein said lymphokine has the properties of: a) increasing neither intracellular calcium ion concentration nor cell membrane potential; b) inhibiting apoptosis by decreasing CD16 expression; c) inducing expression of CD11b d) having no affinity for heparin; and e) having a molecular weight of 50 kd to 80 kd as measured by gel filtration. 2. A method of diagnosing whether a subject is susceptible to drug-induced granulocytopenia, which comprises measuring reactivity of a neutrophil from said subject to GM-CSF or the lymphokine of claim 1. 3. The method of diagnosing according to claim 2, wherein the reactivity of the neutrophil is determined by chemotaxis of or the expression rate of CD11b of the neutrophil as an index of reactivity. |
| PATENT DESCRIPTION |
TECHNICAL FIELD The present invention relates to a novel neutrophil chemotactic lymphokine and a diagnostic agent for diagnosing an attack of drug-induced granulocytopenia comprising this lymphokine. BACKGROUND ART Granulocytopenia refers to a condition that the number of granulocytes in peripheral blood is decreased, and is a concept including agranulocytosis marked by severe decrease or loss in granulocytes and accompanied by a grave condition. Of these, the agranulocytosis has a high mortality due to infectious diseases. As causes of the granulocytopenia, have been known the attack attending on other various diseases, administration of a drug, and the like. Of these, the granulocytopenia caused by the drug includes drug-induced granulocytopenia. The attack of the drug-induced granulocytopenia cannot be predicted prior to the administration of the drug under the circumstances. Further, no drug for treating the drug-induced granulocytopenia after the attack has been known, and so its treatment only depends on stopping the administration of a causative drug under the circumstances. Accordingly, it is an object of the present invention to provide a diagnostic agent capable of predicting an attack of drug-induced granulocytopenia. DISCLOSURE OF THE INVENTION In view of the foregoing circumstances, the present inventors have paid attention to granulocyte chemotactic factors, particularly, neutrophil chemotactic lymphokines and carried out a varied investigation as to their diversity and specificity. As a result, it has been found that a plurality of subspecies different in isoelectric point from one other exist in the neutrophil chemotactic lymphokines. The inventors have succeeded in isolating a novel lymphokine not reported heretofore therefrom. It has also been found that there is a correlation between the chemotaxis of a neutrophil by such a novel lymphokine and the attack of drug-induced granulocytopenia, namely, an individual having a neutrophil low in chemotactic capability to the lymphokine is attacked by drug-induced granulocytopenia, and that the same correlation as described above is also present in a GM-CSF (granulocyte-macrophage colony-stimulating factor), thus leading to completion of the present invention. According to the present invention, there are thus provided a neutrophil chemotactic lymphokine having an isoelectric point at 6.8-7.0 and a cell line which produces such a lymphokine. According to the present invention, there are also provided an agent and a kit for diagnosing drug-induced granulocytopenia, which comprises the lymphokine described above. According to the present invention, there are further provided an agent and a kit for diagnosing drug-induced granulocytopenia, which comprises a GM-CSF. BRIEF DESCRIPTION OF DRAWINGS FIG. 1 diagrammatically illustrates a relationship between isoelectric focusing fractions of CFR-24 culture solution and neutrophil chemotactic activity, FIG. 2 diagrammatically illustrates relationships between chemotaxis indices (CI) of a neutrophil to NCL-4 and GM-CSF and specimens, and FIG. 3 diagrammatically illustrates relationships between expression rates of CD11b in a neutrophil by NCL-4 and GM-CSF and specimens. BEST MODE FOR CARRYING OUT THE INVENTION The neutrophil chemotactic lymphokine (hereinafter referred to as NCL-4) having an isoelectric point at 6.8-7.0 according to the present invention can be obtained from a T cell line obtained by transforming, for example, a normal T cell with a virus. Here, the NCL-4 producing T cell line can be obtained by coculturing, for example, a normal human mononuclear leukocyte together with a human adult T cell leukemia cell line (for example, MT2) and establishing a T cell line transformed with a virus produced by the leukemia cell line. In order to collect NCL-4 from this virus-transformed T cell line, it is only necessary to culture this cell line, conduct isoelectric focusing of the culture solution thereof using neutrophil chemotactic capability as an index and collect a fraction having an isoelectric point of 6.8-7.0. The NCL-4 thus obtained has the following properties: (1) having neutrophil chemotactic capability; (2) having an isoelectric point (pI) of 6.8-7.0; (3) increasing neither intracellular calcium ion concentration nor cell membrane potential; (4) inhibiting apoptosis (decrease in CD16); (5) having the ability to express CD11b; (6) having no affinity for heparin; and (7) having a molecular weight of 50 kd to 80 kd (as measured by gel filtration). The GM-CSF useful in the practice of the present invention is a sort of lymphokine and has been known as a hematopoietic regulatory factor in vivo, but has not been known to be used as a diagnostic agent for drug-induced granulocytopenia. The GM-CSF itself used in the present invention is known, can be prepared in accordance with a method known per se in the art (for example, The Journal of Immunology, Vol. 137, P. 3584 (1986), etc.) and is also commercially available. In order to diagnose drug-induced granulocytopenia using the diagnostic agent according to the present invention, which comprises the NCL-4 or GM-CSF, the reactivity of a neutrophil of a patient, to which a drug will be administered, may be determined in the presence of the diagnostic agent according to the present invention. As a result, it may be diagnosed that the patient has a high possibility of being attacked by drug-induced granulocytopenia if the reactivity of the neutrophil of the patient is low. The reactivity of the neutrophil can be determined or confirmed in accordance with a method known per se in the art. For example, it can be performed by a method making use of the chemotaxis or of the expression rate of CD11b (The Journal of Cell Biology, Vol. 120, No. 2, p. 545 (1993)) of the neutrophil as an index. According to the testing method of neutrophil chemotaxis, the reactivity can be determined in accordance with the conventional means except that the diagnostic agent according to the present invention is used as a chemotactic factor. Examples of such a testing method of neutrophil chemotaxis include a micropore filter method and an agarose method (Respiration, Vol. 4, No. 10, p. 1221 (1985); Journal of Leukocyte Biology, Vol. 51, p.617 (1992), etc.). The result of the test of neutrophil chemotaxis is preferably evaluated, for example, by using chemotaxis index (CI value; chemotactic capability to the diagnostic agent according to present invention/chemotactic capability to a control) as an index. Further, the measurement of the expression rate of CD11b may also be conducted in a method known per se in the art, and can be easily performed by usual immunological analysis or assay making use of a specific antibody thereof. Incidentally, the anti-CD11b antibody can be prepared in accordance with a method known per se in the art or is also commercially available. The object of the diagnostic agent according to the present invention is preferably a patient to which a drug already reported to cause drug-induced granulocytopenia or a drug having the possibility thereof will be administered. No particular limitation is imposed on the preparation form of the diagnostic agent according to the present invention so far as the agent comprises the above-described NCL-4 or GM-CSF. For example, the agent may be provided as a solution with the NCL-4 or GM-CSF dissolved in a buffer solution. The diagnostic agent according to the present invention may also be used as a kit for a clinical test in combination with other reagents, buffers, necessary instruments and the like. Upon diagnosis, the diagnostic agent comprising NCL-4 and the diagnostic agent comprising GM-CSF are separately used to collectively judge the results thus obtained, whereby the diagnosis can be made with higher precision. EXAMPLES The present invention will hereinafter be described in detail by the following Examples. However, the present invention is not limited to these examples. |
| PATENT EXAMPLES | This data is not available for free |
| PATENT PHOTOCOPY | Available on request |
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