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Nucleic acid encoding eosinophil eotaxin receptor

a technology of eosinophil eotaxin and eosinophil eotaxin, which is applied in the field of eosinophil eotaxin receptor, can solve the problems of no reports describing binding studies of any of the -chemokines to primary eosinophils, no ligand selectivity or appropriate expression patterns, etc., and achieves high affinity

Inactive Publication Date: 2007-03-22
DAUGHERTY BRUCE L +3
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0016] The present invention further provides the eosinophil eotaxin receptor, CC CKR3, which is a 13-chemokine receptor and which was cloned from primary eosinophils, and expressed in AML14.3D10 cells. This receptor binds the potent eosinophil attractants, eotaxin, RANTES and MCP-3 with high affinity. In

Problems solved by technology

Although several human β-chemokine receptors have been characterized in detail, none have the appropriate selectivity to account for the observed responses.
Furthermore, there are no reports describing binding studies of any of the β-chemokines to primary eosinophils.
However, neither of these receptors has the necessary ligand selectivity or the appropriate expression patterns required to mediate the effects of the β-chemokines on eosinophils.
In addition, functionality of the receptor was not fully demonstrated.
Fusin has been identified as a cofactor required for infection with virus adapted for growth in transformed T-cells, however, fusin does not promote entry of macrophagetropic viruses which are believed to be the key pathogenic strains of HIV in vivo.

Method used

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Examples

Experimental program
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Effect test

example 1

mRNA Isolation and cDNA Cloning

[0074] Total RNA was isolated from purified eosinophils with TRIzol reagent (BRL) and used in a RT / PCR reaction (Daugherty, B. L., et al. (1991) Nucleic Acids Research 19, 2471-2476) using oligonucleotide primers designed from the human CC CKR1 and MCP-1RB cDNA sequences (Neote, K., et al. (1993) Cell 72, 415-425; Charo, I. F., et al. (1994) Proceecing of the National Academy of Sciences 91, 2752-2756). The primers used for PCR corresponded to a consensus sequence encoded in transmembrane domains (TM) II and VII:

[0075] 5′-PCR primer (TMII) (SEQ ID NO:5):

5′-AACCTGGCCAT(C,T)TCTGA(C,T)CTGC-3′

[0076] 3′-RT / PCR primer (TMVII) (SEQ ID NO:6):

5′-GAAC(C,T)TCTC(C,A)CCAACGAAGGC.

[0077] The resultant PCR product of ˜700 bp was subcloned into plasmid pNoTA (Five Prime, Three Prime, Inc) and sequenced using Sequenase (USB). The remaining 5′ and 3′ sequence encoding CC CKR3 was cloned by rapid amplification of cDNA ends (RACE) using both the 5′-RACE and 3′-RACE ...

example 2

Transfection into AML14.3D10 Human Eosinophilic Cell Line

[0084] AML14.3D10 cells (Paul, C. C., et al. (1995) Blood 86, 3737-3744) were cultured in RPMI-1640, 10% FBS, 1 mM sodium pyruvate, 0.5 μM β-mercaptoethanol and 2 mM L-glutamine (complete medium). Cells were harvested at a density of 0.3×106 / mL, washed once in PBS, resuspended in RPMI at 107 / mL, and 25 μg of plasmid was added. Electroporation was carried out at 300 V, 960 μF using a Gene Pulser (BioRad). Following electroporation, cells were chilled at 0° C. for 10 min and then plated in complete medium at 106 / T75 flask and cultured at 37° C., 5% CO2. After 16-24 hr, cells were pelleted and resuspended in complete medium containing 2 mg / mL Geneticin (GIBCO). Cells were maintained in selection medium for 8-10 days until individual surviving clusters appeared. Individual cells were then transferred to 96-well plates and expanded. AML14 / CCCKR3 sublines were assayed for the ability to generate a Ca2+ flux in response to either R...

example 3

Purification of Eosinophils

[0085] Primary eosinophils were isolated from granulophoresis preparations obtained from allergic and asthmatic donors (Bach, M. K., et al. (1990) Journal of Immunological Methods 130, 277-281). The leukocytes were mixed with equal volumes of HBSS and layered over LSM (Organon Teknika) as described (Rollins, T. E., et al. (1988) Journal of Biological Chemistry 263, 520-526). After lysis of erythrocytes with NH4Cl, the granulocytes were subsequently treated with anti-CD 16 microbeads followed by MACS separation (Miltenyl Biotech) (Hansel, T. T., et al. (1991) Journal of Immunological Methods 145, 105-110). Typically the eosinophil preparations were >99% pure as determined using the LeukoStat staining kit (Fisher).

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PUM

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Abstract

The eosinophil eotaxin receptor has been isolated, cloned and sequenced. This receptor is a human β-chemokine receptor and has been designated “CC CKR3”. The eosinophil eotaxin receptor may be used to screen and identify compounds that bind to the eosinophil eotaxin receptor. Such compounds would be useful in the treatment and prevention of atopic conditions including allergic rhinitis, dermatitis, conjunctivitis, and particularly bronchial asthma.

Description

CROSS REFERENCE TO RELATED APPLICATIONS [0001] This application claims priority under 35 U.S.C. § 119(e) from provisional application Case Number 19634PV, filed Apr. 26, 1996 and from provisional application Case Number 19697PV, filed Apr. 26, 1996 as U.S. Ser. No. 60 / 016,158.FIELD OF THE INVENTION [0002] This invention relates to an eosinophil eotaxin receptor (“CC CKR3”), in particular, the human eosinophil eotaxin receptor and nucleic acids encoding this receptor. This invention further relates to assays which may be used to screen and identify compounds that bind to the eosinophil eotaxin receptor. Such compounds would be useful in the treatment and prevention of atopic conditions including allergic rhinitis, dermatitis, conjunctivitis, and particularly bronchial asthma. BACKGROUND OF THE INVENTION [0003] Eosinophils play prominent roles in a variety of atopic conditions including allergic rhinitis, dermatitis, conjunctivitis, and particularly bronchial asthma (for a reviews see...

Claims

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Application Information

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IPC IPC(8): C12Q1/68G01N33/567C07H21/04C12P21/06C07K14/705A61K38/00A61K51/08C07K14/715C12N15/12
CPCA61K38/00C07K14/7158C12Q2600/158G01N33/6863G01N2333/715C12Q1/6883
Inventor DAUGHERTY, BRUCE L.DEMARTINO, JULIE A.SICILIANO, SALVATORE J.SPRINGER, MARTIN S.
Owner DAUGHERTY BRUCE L
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