Immune stimulation

Claim 2

An immunostimulator factor isolated from bovine thymus which is a peptide having the properties of: A. Strong adherence to Sephadex and BioGel-P at pH 4.5 to 8, B. An isoelectric point of about 7.0 to 7.8, C. Stimulating the DNA-synthetic response of both murine and human lymphocytes to alloantigenic stimulation in a two way MLC, D. Not significantly increasing the stimulation of murine splenic lymphocytes by β-lipopolysaccharides, E. Ability to enhance immunostimulatory response even in the presence of 2-mercaptoethanol, F. Totally reversible in MLC if removed up to 72 hours from the start of MLC, G. Functioning more effectively in MLC containing 10% human serum than in MLC medium containing 10% fetal calf serum, H. Ability to enhance immunostimulatory response in MLC even in presence of mouse serum, I. Ability to accelerate the rejection of histoincompatible skin grafts in mice, J. Ability to pass through dialysis tubing with a 3500 Dalton cut-off, K. Splitting into two immunostimulatory fractions, one with a molecular weight of about 1400 Daltons and the other less than 1400 Daltons on gel filtration at pH 10.2 in 0.05M ammonium hydroxide.

Claim 3

A process for the preparation of an immunostimulatory factor from bovine thymus comprising the steps of: A. Extracting thymus tissue with aqueous buffer at pH 8.5 at about 0° C. to 5° C., B. Centrifuge homogenate at 26,000 to 30,000×g for 20 to 40 minutes at about 0° C. to 5° C., C. Reextract pellet from Step B as in Step A and centrifuge homogenate as in Step B, D. Pool supernatants from Steps B and C and lyophilize, E. Suspend lyophilized powder in water at about 0° to 5° C. at a concentration of from 0.25 to 0.3 gm/ml and slowly add absolute ethanol with stirring to a final concentration of 50% to 60% (V/V), F. Stir for 16 to 18 hours at 0° to 5° C. and centrifuge at 26,000 to 30,000×g for 20 to 40 minutes, G. Add supernatant to 5 to 6 volumes of acetone at about -10° C. to -5° C. and let stand for 3 to 6 days at about -10° C. to -5° C., H. Separate precipitate, suspend in about 100 to 200 ml of distilled water or the same amount of about 20 mM acetic acid and lyophilize at 0° to 5° C., I. Dissolve lyophilized powder in buffer at pH 4.5 to 8 and separate over Sephadex or BioGel equilibrated wih the same buffer, J. Collect fractions and assay by MLC to select the fractions with immunostimulatory activity.

Claim 4

A process for the preparation of an immunostimulatory factor from bovine thymus comprising the steps of: A. Extracting thymus tissue with aqueous buffer at pH 8.5 at about 0° C. to 5° C., B. Centrifuge homogenate at 26,000 to 30,000×g for 20 to 40 minutes at about 0° C. to 5° C., C. Reextract pellet from Step B as in Step A and centrifuge homogenate as in Step B, D. Pool supernatants from Steps B and C and lyophilize, E. Suspend lyophilized powder in water at about 0° to 5° C. at a concentration of from 0.25 to 0.3 gm/ml and slowly add absolute ethanol with stirring to a final concentration of 50% to 60% (V/V), F. Stir for 16 to 18 hours at 0° to 5° C. and centrifuge at 26,000 to 30,000×g for 20 to 40 minutes, G. Add supernatant to 5 to 6 volumes of acetone at about -10° C. to -5° C. and let stand for 3 to 6 days at about -10° C. to -5° C., H. Separate precipitate, suspend in about 100 to 200 ml of distilled water or the same amount of about 20 mM acetic acid and lyophilize at 0° to 5° C., I. Dissolve lyophilized powder in buffer at pH 4.5 to 8 and separate over Sephadex or BioGel equilibrated wih the same buffer, J. Collect fractions and assay by MLC to select the fractions with immunostimulatory activity.

Claim 5

A pharmaceutical composition comprising a pharmaceutically acceptable carrier and an amount which is effective to generate an immunostimulatory response of an immunostimulatory factor isolated from bovine thymus which is a peptide having the properties of: A. Strong adherence to Sephadex and BioGel-P at pH 4.5 to 8, B. An isoelectric point of from 7.0 to 7.8, C. Stimulating the DNA-synthetic response of both murine and human lymphocytes to alloantigenic stimulation in a two way MLC, D. Not significantly increasing the stimulation of murine splenic lymphocytes by β-lipopolysaccharides, E. Ability to enhance immunostimulatory response in the presence of 2-mercaptoethanol, F. Totally reversible in MLC if removed up to 72 hours from the start of MLC, G. Functioning more effectively in MLC containing 10% human serum than in MLC medium containing 10% fetal calf serum, H. Ability to enhance immunostimulatory response in MLC even in presence of mouse serum, I. Ability to accelerate the rejection of histoincompatable skin grafts in mice, J. Ability to pass through dialysis tubing with a 3500 Dalton cut-off, K. Splitting into two immunostimulatory fractions, one with a molecular weight of about 1400 Daltons and the other less than 1400 Daltons on gel filtration at pH 10.2 in 0.05M ammonium hydroxide.

Claim 6

A pharmaceutical composition comprising a pharmaceutically acceptable carrier and an amount which is effective to generate an immunostimulatory response of an immunostimulatory factor isolated from bovine thymus which is a peptide having the properties of: A. Strong adherence to Sephadex and BioGel-P at pH 4.5 to 8, B. An isoelectric point of from 7.0 to 7.8, C. Stimulating the DNA-synthetic response of both murine and human lymphocytes to alloantigenic stimulation in a two way MLC, D. Not significantly increasing the stimulation of murine splenic lymphocytes by β-lipopolysaccharides, E. Ability to enhance immunostimulatory response in the presence of 2-mercaptoethanol, F. Totally reversible in MLC if removed up to 72 hours from the start of MLC, G. Functioning more effectively in MLC containing 10% human serum than in MLC medium containing 10% fetal calf serum, H. Ability to enhance immunostimulatory response in MLC even in presence of mouse serum, I. Ability to accelerate the rejection of histoincompatable skin grafts in mice, J. Ability to pass through dialysis tubing with a 3500 Dalton cut-off, K. Splitting into two immunostimulatory fractions, one with a molecular weight of about 1400 Daltons and the other less than 1400 Daltons on gel filtration at pH 10.2 in 0.05M ammonium hydroxide.

Claim 7

A pharmaceutical composition of claim 3 in dosage unit form.