John E. McClure

Current Status of Thymosin and Other Hormones of the Thymus Gland

Publisher Summary This chapter discusses the current status of the chemistry, biology, and clinical applications of the well-defined thymic hormones. The first biologically active polypeptide to be isolated from among the highly acidic components of bovine thymosin fraction 5 has been termed thymosin α1. This peptide is highly active in amplifying T-cell immunity and is active in modulating the expression of terminal deoxynucleotidyl transferase. The ultimate application of the thymosins and other thymic hormones and factors in cancer treatment should be in providing a means of safely augmenting specific T lymphocyte functions in patients with diminished thymic-dependent immunity. In anergic cancer patients, thymic hormones may be of importance as an adjunct to conventional treatments by increasing T-cell function in response not only to tumor cells but also to pathogens, thus reducing the high incidence of infection that often accompanies cancer treatment.

Current status of thymosin research: evidence for the existence of a family of thymic factors that control T-cell maturation*..

Thymosin fraction 5 contains several distinct hormonal-like factors which are effective in partially or fully inducing and maintaining immune function. Several of the peptide components of fraction 5 have been purified, sequenced and studied in assay systems designed to measure T-cell differentiation and function. These studied indicate that a number of the purified peptides act on different subpopulations of T-cells (see Figure 1). Thymosin beta 3 and beta 4 peptides act on terminal deoxynucleotidyl transferase (TdT) negative precursor T-cells to induce TdT positive cells. Thymosin alpha 1 induces the formation of functional helper cells and conversion of Lyt- cells to Lyt 1+, 2+, 3+ cells. Thymosin alpha 7 induces the formation of functional suppressor T-cells and also converts Lyt- cells to Lyt 1+, 2+, 3+ cells. These studies have provided further evidence that the thymus secretes a family of distinct peptides which act at various sites of the maturation sequence of T-cells to induce and maintain immune function. Phase I and Phase II clinical studied with thymosin in the treatment of primary immunodeficiency diseases, autoimmune diseases, and cancer point to a major role of the endocrine thymus in the maintenance of immune balance and in the treatment of diseases characterized by thymic malfunction. It is becoming increasingly clear that immunological maturation is a process involving a complex number of steps and that a single factor initiating a single cellular event might not be reflected in any meaningful immune reconstitution unless it is the only peptide lacking. Given the complexity of the maturation sequence of T-cells and the increasing numbers of T-cell subpopulations that are being identified, it would be surprising if a single thymic factor could control all of the steps and populations involved. Rather, it would appear that the control of T-cell maturation and function involves a complex number of thymic-specific factors and other molecules that rigidly control the intermediary steps in the differentiation process.

Effects of 6-hydroxydopamine upon primary and secondary thymus dependent immune responses.

Adult male mice were treated with various doses of 6-hydroxydopamine in order to assess the effects of this drug upon thymic dependent immunity. A consistent decrease in primary antibody titers to sheep erythrocytes was observed following treatment with this drug. Serum levels of thymosin alpha 1 were increased by day three after 6-OHDA with a return to normal by day five. Thymocyte terminal deoxynucleotidyl transferase changes were biphasic with an initial decrease after 6-OHDA followed by an increase. Changes in mitogen responsiveness were observed but were not consistently reproducible. Involvement of both catecholamines and corticosteroids in bringing about these observed changes was discussed.

Immunochemical studies on thymosin: Radioimmunoassay of thymosin β4

Thymosin beta 4, a peptide with hormonal-like properties first isolated from the thymus gland, can be measured in serum using a newly described radioimmunoassay. The radioimmunoassay utilizes an antibody raised in rabbits against synthetic thymosin beta 4 conjugated by glutaraldehyde to keyhole limpet hemocyanin. A 125I-tyrosine-C13 analogue of the biologically active C-terminal fragment is used as the radioactive tracer. The radioimmunoassay is sensitive in the nanogram range and no cross-reactivity with common serum proteins is demonstrable. High performance liquid chromatography of serum samples indicates that two thymosin beta 4 cross-reactive species are present in human serum. Levels in serum range from 450 to 1100 ng/ml and decline with age.

Immunocytochemical localization of thymosin-α1 in thymic epithelial cells of normal and myasthenia gravis patients and in thymic cultures☆

Thymosin alpha 1 (alpha 1) is a potent thymic polypeptide hormone. With antibodies against synthetic thymosin alpha 1, indirect immunofluorescence was applied to human normal thymus and to hyperplastic, thymomatous or involuted thymus of myasthenia gravis (MG) patients. Alpha 1 was localized only in the epithelial cells, lying singly, grouped, in Hassalls corpuscles or proliferated in thymomas. In contrast to normal thymus, which had fewer and more weakly stained cells, MG hyperplastic thymus had many strongly positive epithelial cells: this was markedly evident in thymomas. Involuted MG thymus had a few but brightly stained cells lying within the fatty tissue. In tissue cultures of human thymus, anti-alpha 1 stained the epithelial cells, but not fibroblasts. These findings: (a) demonstrate the origin of the thymic hormone alpha 1 to be the thymic epithelial cell; (b) raise the possibility that excess alpha 1 may act pathologically to facilitate and perpetuate the dysimmune mechanism in MG; (c) may partially explain the beneficial effect of thymectomy in MG patients of any age; and (d) suggest that epithelial cells may be autonomous for the production of alpha 1 as evidenced by their positivity in tissue culture.

Isolation of thymosin α1 from thymosin fraction 5 of different species by high-performance liquid chromatography

High-performance liquid chromatography (muBondapak C18 column with 0.05% trifluoroacetic acid in acetonitrile as solvent system) was used to isolate thymosin alpha 1 (alpha 1) from thymosin fraction 5 (f5) of various species (calf, pig, sheep and mouse). Each of the f5 preparations gave a protein peak similar in retention time to bovine thymosin alpha 1. This peak coincided with the immunoreactive peak determined by a radioimmunoassay for alpha 1. Chromatographic analysis of fresh thymus tissue extracts using a high-performance liquid chromatographic similar system did not reveal a detectable protein peak or immunoreactive peak at the alpha 1 position. Our results suggest that alpha 1 may be synthesized in a precursor form in animal tissues.

Presence of thymosin-like factors in human thymic epithelium conditioned medium

The objective of the present study was to determine whether or not thymosin and conditioned medium from human thymus epithelial cultures (HTECM) contain similar fractions, capable of inducing T-cell differentiation. Therefore we tested the ability of rabbit antisera to different thymosin fractions (thymosin fractions 5, 6 and alpha 1) of both bovine and human origin to block the stimulatory effect of HTECM on Con A and PHA response of mouse thymocytes. We also looked for reactivity of the antisera toward tissues of man, mouse and calf, and to tissue cultures of man and mouse. Anti-thymosin fraction 5 and 6, but not anti-thymosin-alpha 1, were found to inhibit the stimulatory effect of HTECM on both Con A and PHA responses. This cannot be attributed to cytostatic or cytotoxic effects of the antisera on thymocytes. No effect was seen when using antiserum to kidney fraction 5 or normal rabbit serum. In both tissue sections and tissue cultures of the different species tested, anti-thymosin reacts only with thymus epithelial cells. The reactivity is blocked by neutralizing the antisera with thymosin fraction 5 but not by kidney fraction 5. The reactivity is also strongly diminished by neutralizing the antisera with HTECM but not with supernatants of conditioned media from non-thymic tissues. The observations are highly suggestive for the presence of similar fractions in HTECM and thymosin, and that both are secreted by thymus epithelial cells.

IDENTIFICATION OF HUMAN THYMIC EPITHELIAL CELLS WITH ANTIBODIES TO THYMOSIN α1, IN MYASTHENIA GRAVIS*

Thymosin-alpha 1 (alpha 1) is a potent thymic polypeptide hormone. Using anti-alpha 1 antibodies, we applied indirect immunofluorescence to human normal thymus of different ages and to hyperplastic, thymomatous, and involuted thymus of myasthenia gravis (MG) patients. Alpha 1 was localized only in the epithelial cells, lying singly, grouped, in Hassells corpuscles, and proliferated in thymomas. Whereas normal thymuses and fewer and weakly stained cells, MG thymuses had many strongly positive epithelial cells; this was more evident in thymomas. Germinal centers were unstained. Involuted MG thymuses had small islands of brightly stained cells lying among the fatty tissue. In cultured thymuses from three MG patients, epithelial cells but not fibroblasts were brightly stained for alpha 1. Our findings (a) demonstrate the location, and presumably the origin, of alpha 1 to be the thymic epithelial cell; (b) suggest the possibility that excess alpha 1, because of its known effect on T-lymphocyte maturation and transformation of precursors to helper T-cells, may act pathologically to facilitate and perpetuate the dysimmune mechanism in MG; (c) may partially explain the beneficial effect of thymectomy in MG patients of any age; and (d) indicate that epithelial cells may be autonomous for the production of alpha 1 as evidenced by their alpha 1 positivity in culture.

Radioimmunoassay of thymosin beta4

A radioimmunoassay for the polypeptide thymic hormone thymosin beta 4 is described. The assay employs a radiolabelled Tyr-C13-thymosin beta 4-analogue as probe and a thymosin beta 4 antibody.

An Evaluation of Two Different Schedules of Synthetic Thymosin α1 Administration in Patients with Lung Cancer

Thymosin α1 (molecular weight 3108) is one of the many active polypeptides isolated from thymosin fraction 5 (TF5) (Low and Goldstein, 1979; Goldstein et al., 1982). α1 was initially purified from extracts of calf thymus glands. Biologically active α1 has now been successfully synthesized by classical solution (Birr and Stollenwerk, 1979; Wang et al., 1978), solid-phase (Wang et al., 1980; Folkers, et al., 1980) and recombinant DNA procedures (Wetzel et al., 1980).