Method of increasing immunologic competence

#The thymus has been recognized as an endocrine gland having a major function in the immunological defense system of the body. Recently, crude extracts of calf thymus have been shown to contain a family of hormones, referred to as thymosins, having molecular weights ranging from approximately 3,200 to 70,000 daltons. See for example, Trainin, N., Physiological Reviews, Vol. 54, p. 272 (1974) and White, A., Ann. N.Y. Acad. Sci., Vol. 29, p. 253 (1975).

#These hormones are believed to act by regulating the rate of maturation of incompentent, as yet unidentified, precursor cells to competent lymphocytes (T-cells) and thus to become effective fighters against foreign invaders. In addition, precursor cells of other types, e.g., erythroid, are stimulated in their production and maturation by thymosin preparations. Recently, a relatively crude calf thymus extract containing thymosin was used to increase the immunologic competence of a young girl born with impaired immunity. Wara, D.W., et al., New Eng. J. Med., Vol. 292, p. 70 (1975). Also a recent report has described improvement in the total hematological status (increase in number of both white and red cells) in patients treated with a thymosin preparation. See Alexsandrowicz, J., et al., Proc. Intl. Congress Immunol., Brighton, England, July 22-24, 1974.

#There have also been reports, see for example Bach, J. F., et al., Immunology, Vol. 25, p. 353 (1973), that thymosin-like activity has been detected in fresh pig blood. The factor responsible therefor is believed to have a molecular weight of approximately 1,000 daltons.

#Four kilos of Cohn human blood fraction IV-1 (Cutter Laboratories) were stirred in 20 l. of double distilled water at 5.degree. C. for four hours and lyophilized to afford 1105 g. of dry material which was suspended in ten volumes of deionized water. The pH was adjusted to 7.0 with ammonium hydroxide and the mixture stirred for one hour at room temperature. The sediment was removed and the supernate passed through an HIOX-50 cartridge in an Amicon DC-30 apparatus. The lower molecular weight fraction from this step was then passed through an HIOSM cartridge in the same apparatus. After lyophilization of the retentate there was obtained 50 g. of material which was chromatographed, in one gram portions, (after dissolution in the below-mentioned buffer) on a 5 .times. 90 cm. G-75 Sephadex (Phamacia Fine Chemicals) column equilibrated with 50 mM Tris-100 mM NaCl-0.02% sodium azide buffer, pH 8.0, at 5.degree. C. The chromatography was monitored by optical density measurements at 280 nm, the contents of each tube were lyophilized and diafiltered to determine total protein, and then subjected to an in vitro rosette assay. Over 90% of the activity eluted was recovered in a fraction slightly retarded on this column with a K.sub.av value of 0.055, corresponding to an approximate molecular weight range of about 50,000 to 70,000 daltons. This material (total from all one gram runs = 20 g.) was then subjected to free-flow electrophoresis at pH 5.25 in an acetate buffer system (25 mM NaOAc - 7 mM HOAc). As above, the progress was monitored by optical density, total protein and bioassay. The material resulting from combination of active fractions was further purified by preparative polyacrylamide gel electrophoresis using a 10% cross-linked gel. The spacer and sample gels were 0.060 M in Tris, adjusted to pH 6.6-6.8 with HCl. The separator gel was 0.375 M in Tris, adjusted to pH 8.9 with HCl. THe electrode vessel buffer was 0.024 M in Tris and 0.192 M in glycine, pH 8.2-8.4. The elution buffer was 0.375 M in Tris, adjusted to pH 8.8-9.0 with HCl. The activity was eluted in a fraction migrating between the tracking dye (Bromophenol Blue) and the albumin fraction. This material (approximately 100 mg. total) was electrophoretically homogeneous when run in an analytical system at pH 8.9.

#Repeat of the Sephadex chromatography, as described above, on a micro (0.8 .times. 76 cm.) column afforded essentially pure prealbumin as a white solid.

#The S.sub.20w value determined by sedimentation velocity in the Spinco model E ultracentrifuge was found to be 4.07 and the molecular weight of the molecule determined by sedimentation equilibrium was found to be 56,700 daltons. After treatment with 5M guanidinium-hydrochloride - 0.8% dithiothreitol -0.01M phosphate buffer pH 5.2 followed by 5M urea - 0.1% sodium dodecyl sulfate - 1% dithiothreitol -0.01M phosphate buffer pH 7.4 followed by electrophoresis at pH 7.0 in a sodium dodecylsulfate - containing acrylamide gel, the molecular weight of the undissociated material was found to be 52,000 daltons and there was partial dissociation into four sub-units with a molecular weight of approximately 13,500 daltons each.