Purification and Biological Activity of Thymosin, a Hormone of the Thymus Gland

Abstract

The purification and chemical properties of thymosin, obtained from bovine thymus tissue, are described. The biological activity of the thymic hormone has been assessed by a newly developed rosette assay, which permits measurement of thymus-dependent lymphoid cells. Thymosin activity is associated with a physicochemically homogeneous protein of molecular weight 12,600. The hormonal activity is evident in in vitro incubation assay, after injection into adult thymectomized mice, and in prolonging survival of neonatally thymectomized mice and the reconstitution of their response to a skin allograft.The extraction, partial purification, and assay of a thymic factor, which has been termed thymosin, from calf thymic tissue has been described by this laboratory (1). Thymosin-containing fractions lowered the incidence, in neonatally thymectomized mice, of a wasting disease characterized by an atrophy of lymphoid tissue and high mortality (2) and a failure of development of cell-mediated immune responses, including the capacity of host cells to elicit a normal graftversus-host response (3) and to reject histoincompatible skin grafts (4). In addition, thymosin administration to newborn normal mice accelerated the ontogenesis of cell-mediated immunity, as measured by the capacity of spleen cells from the treated animals to elicit a normal graft-versus-host response (5) and by the development of resistance to progressive tumor growth after inoculation with Moloney sarcoma virus (6). The addition of thymosin preparations in vitro to discrete populations of lymphoid stem cells from bone marrow rapidly converted such cells into immunologically competent cells, as measured by a graft-versus-host assay (5) and by the appearance of cells with characteristics of T-cells (7) in the rosette bioassay.In this preliminary communication, we describe the further purification and characterization of thymosin. We have isolated from thymus glands of calves a carbohydrate-and lipid-free homogeneous protein that, in preliminary assays, has several of the biological activities that we have reported for crude fractions (8,9). The purification and activity are based upon a modification of a new rosette-forming cell assay described recently (7). A complete paper on the chemistry and additional properties of thymosin will be reported in a separate communication (Guha, A. et MATERIALS AND METHODSAnimals. CBA/J and A/J male mice, 60 days old, were purchased from Jackson Laboratories, Bar Harbor, Me. CBA/ Wh mice of the same age were raised in our own colony. All animals received food and water ad libitum until killed.Chemicals and Reagents. Azathioprine was generously provided by Burroughs-Wellcome as its sodium salt. Sephadex G-150 (40-120 ,um) was purchased from Pharmacia Chemicals, Inc.; Cellex E (Ecteola; 0.43 meq/g exchange capacity) and Biogel HTP were obtained from Bio-Rad Laboratories. All other chemicals were of analytical or reagent grade and were used without further purification.Rosette Assays. Two types of rosette assays for ...

Binding of autologous erythrocytes to immature T-cells.

“…This was prepared by injecting 107 CBA thymocytes intraperitoneally (i.p.) into AKR mice once weekly for 8 …”

“…Thymosin (fraction V) was obtained from A. L. Goldstein (8). Thymic humoral factor (THF), a calf thymus dialysate, was from N. Trainin (9).…”

Effect of Thymic Humoral Factor on Cellular Immune Functions of Normal Children and of Pediatric Patients with Ataxia Telangiectasia and Down's Syndrome

“…A variety of factors of thymic origin has been isolated (9,25,26) and found to enhance maturation of such precursors (1,3,19,20,26,32,34).…”

The Level of Metabolic and Immunological Status of Newborn Calves Under the Action of Timogen on the Body of Down-calving Cows

“…Glutamic acid, which is part of it, belongs to the group of amino acids, able to accelerate the differentiation of precursors of Tcells into T-lymphocytes and to amplify the response to heterologous erythrocytes in vitro on the splenocytes that was revealed in the experiments with laboratory animals. Timogen stimulated humoral immune response in thymusdeprived mice, increased the contents of cyclic nucleotides in splenocytes, as well as the chemotaxis and phagocytosis of neutrophils, stimulated liver regeneration in rats after partial hepatectomy and in experimental cirrhosis [12][13][14][15] .…”

“…It is known that the timogen increases the immunological reactivity of the organism, strengthens the lymphoid cells differentiation processes, normalizes the number of T-helpers, Tsuppressors and their ratio in blood and lymphoid organs, as well as stimulates cellular metabolism and increases the animal growth rate [12][13][14] . The drug is prescribed in cases of immunodeficiencies, disorders of regenerative processes, for the prevention of viral and bacterial infections, to increase the immune response and animal growth rate 10,15 .…”