Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Lymphokines – e.g. – interferons – interlukins – etc.
Patent
1984-09-26
1986-09-23
Kight, John
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Lymphokines, e.g., interferons, interlukins, etc.
435 68, 435240, 424 95, C07K 1506
Patent
active
046134581
DESCRIPTION:
BRIEF SUMMARY
TECHNICAL FIELD
The present invention relates to a novel regulatory substance of T cell DNA synthesis. More specifically, the present invention relates to regulatory substances of T cell DNA synthesis obtained by culturing fibroblasts or lymphocytes of animals or insects in a medium free of serum or containing bovine fetal serum or by recovering from the serum of animals or body fluids of insects.
BACKGROUND ART
Heretofore, steroids, immuran, and the like have been used for the treatment of various autoimmune diseases caused by unusual acceleration of immune activity in immunocompetent cells such as T cells. Although these drugs have considerable therapeutic effects upon certain diseases, they have side-effects such as adrenal insufficiency and leukopenia against normal cells. Therefore, more excellent drugs are still in demand.
The present inventors have found that fibroblasts and lymphocytes of animals produce a substance which regulates the DNA synthesis of immunocompetent cells such as T cells and that the substance regulates the activity of immunocompetent cells and is useful for the treatment of autoimmune diseases. Thus the present invention has been completed
Regulatory substances against immunocompetent cells have been reported in J. Immunol 109, 154-163 (1972), J. Immunol 116, 1452-1458 (1976) and Inflammation 1, 5-21 (1975). Since these known substances are high molecular substances, they have possibility of bringing about side-effects such as immuno-refusal effect of patients. The substance of the present invention is low molecular peptide derived from mammals and therfore, is expected to produce less side-effect and to be applicable to immuno-diseases.
DISCLOSURE OF THE INVENTION
The novel regulatory substance of T cell DNA synthesis of the present invention (referred to as "the present substance" hereinafter) is obtained by culturing fibroblasts or lymphocytes of animals or insects in a medium and recovering the present substance accumulated in the culture medium.
As the animal, mammals such as mouse, rat, human and the like are preferably used. As the cells, mouse C3H/He spleen cells, mouse 3T3 fibroblasts [J. Cell Biol., 17, 299 (1963)], rat spleen cells SD (Splague Dawley) and human fetal fibroblasts are preferably used. These cells can be obtained from Seiwa Jikken Dobutsu Co., Ltd., Ohita.
As the cells of insects, fibroblasts established from Antheraea eukarypti [Grace, Nature, 195, 788 (1962)] are preferably used.
As the medium, any medium employable for the culture of animal cells may be used. Preferably, RPMI 1640 medium, MEM medium, and the like are used. The compositions of RPMI 1640 medium and MEM medium are described in "Soshiki Baiyo (Tissue Culture)" edited by Junnosuke Nakarai, et al., published by Asakura Shoten, pages 9-11, 1976. 1-10% bovine fetal serum may be added to the medium. Addition of phorbol esters to the medium increases the productivity of the present substance.
As the phorbol esters, phorbol myristate, phorbol-13acetate and 12-O-tetradecanoyl-phorbol-13-acetate (TPA) are preferably used.
The phorbol esters are generally used in a concentration of 1-1,000 ng, preferably 10-100 ng per 1 ml of culture medium.
Culturing is carried out under aeration in a liquid medium according to conventional culture conditions for animal cells. 5-10% CO.sub.2 and 90-95% air are aerated. Culturing temperature is 25.degree.-40.degree. C., preferably 30.degree.-37.degree. C., pH is 7.5-8.5, preferably 7.8-8.0 and culturing time is preferably 5-24 hours. Recovery of the regulatory substance of T cell DNA synthesis from culture medium is carried out according to a conventional method used for recovering proteins from culture medium of animal cells.
After culturing, cells are removed by centrifugation to recover the culture supernatant. Twice as much saturated ammonium sulfate (pH 7.5) as the supernatant by volume is added thereto and subjected to salting-out. The salting-out material is centrifuged and the resulting precipitate is dissolved in a physiological saline solution. The
REFERENCES:
Regulatory Serum . . . Lipo Protein, J. Immunology 116(5) 1976, p. 1452, Curtis et al.
The Effect of Immunogulation . . . in vitro, J. Immunol 109(1) 1972, p. 154, Cooperband et al.
C.A. No. 21197p vol. 101, 1984 DNA Synthesis Inhibitor Block Protein Synthesis . . . , Lymphocyte, Albert et al.
C.A. No. 154526w, vol. 100, 1984, Purifed Plasma . . . 3T3 Cells, Vale et al.
C.A. No. 20457g, vol. 92, 1979, Normal Immunosuppressive Protein . . . Cell Line, Ovodia et al.
Draper Garnette D.
Kight John
Kyowa Hakko Kogyo Co. Ltd.
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