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Method and cell composition for screening compounds for anti-inflammatory activity

a technology of anti-inflammatory activity and cell composition, which is applied in the field of method, cell composition and assay for screening compounds for anti-inflammatory activity in vitro, can solve the problems of eventual mortality of the host and morbid states

Inactive Publication Date: 2005-09-22
GENETROL BIOTHERAPEUTICS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Benefits of technology

[0029] In another aspect, the invention provides an in vitro method for screening test compounds for anti-inflammatory activity. In practicing the method, the above cells are cultured under conditions in which a cytokine regulatory factor is overproduced and cytokine production is stimulated, such that the detectable-marker protein, encoded by a cDNA dri

Problems solved by technology

However, overproduction of proinflammatory cytokines can result in extensive damage to host tissues, leading to morbid states, and in severe cases, eventual mortality of the host.

Method used

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  • Method and cell composition for screening compounds for anti-inflammatory activity
  • Method and cell composition for screening compounds for anti-inflammatory activity
  • Method and cell composition for screening compounds for anti-inflammatory activity

Examples

Experimental program
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example 1

Preparation of a pcCMV-PKR Vector and a PKR(−) Control Vector

[0148] cDNA encoding the full-length human PKR molecule (551 amino acids; Meurs, et al., 1990; GenBank Accession No. NM002759) was inserted into a eukaryotic expression vector, such that the PKR coding sequence was expressed under the control of the CMV promoter.

[0149] The vector contains various features suitable for PKR transcription, including: i) a promoter sequence from the immediate early gene of the human CMV for high level mRNA expression; ii) a polyadenylation signal and transcription termination sequences from the β-globin gene to enhance RNA stability; iii) an ampicillin resistance gene and ColE1 origin for selection and maintenance in E. coli.

[0150] A second vector contained i) an ampicillin resistance gene and a ColE1 origin for selection and maintenance in E. coli; ii) the G418 resistance marker (Neo) to allow for selection and identification of the plasmids after co-transfection into eukaryotic cells.

example 2

Preparation and Analysis of a PKR Over-producing Namalwa Cell Line Over-expressing Inflammatory Cytokines

[0151] Stable transfectants were obtained by electroporation of 4×106 exponentially growing Namalwa cells with 15 μg of the vector pCMV-PKR and 15 μg of the Neo containing second vector in DMEM / F12 (+10% FBS) using a Gene Pulser apparatus (BioRad) set at 800 μF, 300 V. Bulk populations of stable transformants were obtained by selection with 2 mg / ml geneticin (Gibco-BRL) for 3-4 weeks. Clonal lines were subsequently obtained by limiting dilution cloning.

[0152] A. Increased Inflammatory Cytokine Expression Following Induction

[0153] The level of PKR in the parental and PKR-transfected Namalwa cells were analyzed by Western blot and PKR activity assay (Zamanian-Daroush, et al., 1999). PKR expression was found to have increased approximately sixteen-fold in the PKR-transfectants relative to the parental Namalwa cells.

[0154] PKR-transfected Namalwa cells (Nam-PKR) and parental Nama...

example 3

Suppression of PKR-mediated Inflammatory Cytokine Production by a PKR-Specific Inhibitor and Potential Anti-inflammatory Drug

[0156] A. Suppression of PKR-mediated Inflammatory Cytokine Production by 2 Aminopurine

[0157] PKR-transfected Namalwa cells (Nam-PKR) were cultured at 2.5×105 cells / ml in DMEM / F12 medium supplemented with 10% FBS. The cells were treated with 20 mM PMA (priming) for 20 hr followed by treatment with 200 μg / ml poly I:C (induction) for 3 days in the presence of 1 mM 2-aminopurine (2-AP) or in the absence of 2-AP. One set of cells was left untreated (non-induced controls). Following treatment, the culture supernatants were collected and analyzed for TNF-alpha or for TNF-beta by ELISA according to the procedure provided by the supplier of the ELISA kits (R&D Systems). The data for cytokine induction is shown in FIGS. 2A and 2B, which illustrates that the PKR-overexpressing Namalwa cell line produces a high level of proinflammatory cytokine (TNF-alpha) under induct...

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Abstract

A method and cell line for screening test compounds for antiinflammatory activity are disclosed. The cell line is a human cell line capable of producing a selected cytokine associated with an inflammatory response in humans, and transfected with (i) a vector containing DNA encoding a cytokine regulatory factor under the control of a first promoter, and (ii) a vector containing DNA encoding a detectable-marker protein, under the control of a second promoter which is responsive to cytokine induction. In the screening method, the cells are cultured under conditions of cytokine regulatory factor overexpression and cytokine induction. Addition of test compound that results in a diminution of the detectable-marker protein is evidence of anti-inflammatory activity.

Description

[0001] This application is a continuation of U.S. patent application Ser. No. 09 / 952,571, filed Sep. 13, 2001, which claims priority to U.S. Provisional Patent Application Ser. No. 60 / 232,385 filed on Sep. 14, 2000, which is incorporated herein in its entirety by reference.FIELD OF THE INVENTION [0002] The present invention relates to a method, cell composition and assay for screening compounds for anti-inflammatory activity in vitro by suppressing cytokine regulatory factor-mediated induction of proinflammatory cytokines. REFERENCES [0003] Abbas, A. K., et al, Eds., Cellular and Molecular Immunology, 4th edition, WB Saunders Co., 246-415, 2000. [0004] Abraham, E., Critical Care Med, 28: 100-104, 2000. [0005] Baldwin, A. S., Ann. Rev. Immunol., 14: 649-681, 1996. [0006] Barone, F. C. and Feuerstein, G. Z., J. Cereb. Blood Flow Metab., 19:819-834, 1999. [0007] Christman, J. W., et al., Chest, 117: 1482-1487, 2000. [0008] Goh, K. C., et al., EMBO, [0009] Handel, M. L., et al., Clin Ex...

Claims

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Application Information

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IPC IPC(8): C12N5/00C12N5/02C12N5/10C12N15/12C12N15/63C12N15/85C12N15/87C12P19/28C12P21/06C12Q1/00C12Q1/68G01N33/53G01N33/567
CPCC12N15/63C12N15/85C12N2503/02C12N2800/108G01N2333/54C12N2830/85C12N2840/20G01N2333/52C12N2830/00
Inventor LAU, ALLAN S.KIEFER, MICHAEL C.
Owner GENETROL BIOTHERAPEUTICS
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