Distribution of thymic hormones in thymic tumors and myasthenic thymus.

To understand the thymic hormone production in thymic tumors and the myasthenic thymus, we have studied the concentrations of two thymic hormones (prothmosin alpha and thymosin beta 4) in these tissues. A total of forty four thymus or thymic tumor tissues were obtained from patients who underwent thoracic operation. These tissues consisted of a control group (n = 25) and three study groups including a thymoma group (N = 8), thymic carcinoma group (n = 3) and myasthenic thymus group (n = 8). The age-related curve of the control group, which provided histologically normal thymic tissues for the production of prothymosin alpha and thymosin beta 4, was established. It was found that in the control group the concentrations (microgram/g tissue) of prothymosin alpha and thymosin beta 4 decreased with age, and that the ranges were from 171 micrograms/g to trace and from 243 micrograms/g to trace, respectively. The amounts were highest at puberty and then gradually decreased, and only trace amounts of both polypeptides were present at age 60. Study cases which deviated from the standard curves were identified. Our results indicated that the contents of thymic hormones significantly increased in the tissues of invasive or non-invasive thymoma, decreased in thymic carcinoma, and were not significantly changed in myasthenic thymus as compared with those of the age-related normal thymus tissues. These findings confirm the notion that only thymomas produce significantly higher thymic hormones.

In vivo induction of apoptosis in immature thymocytes by staphylococcal enterotoxin B.

Staphylococcal enterotoxins are potent T cell mitogens. Recent studies have shown that the binding of these toxins to class II MHC molecules on accessory cells is essential for the stimulation of T cells which bear specific V beta segment of TCR. In the present study we show that i.v. administration of staphylococcal enterotoxin B (SEB) results in an enlargement of spleen and lymph nodes but causes thymus atrophy. Elimination of CD4+CD8+ cells predominantly accounted for the shrinkage of thymus, and the lowest level of this cell population was reached 4 days after SEB injection. Furthermore, this decrease in CD4+CD8+ cells was accompanied by a relative increase in the percentages of CD4+CD8-, CD4-CD8+ and CD4-CD8- cells, whereas their absolute numbers actually reduced on day 4. The thymus shrinkage involved apoptosis which was characterized by DNA fragmentation and morphologic changes. The depletion of Thy-1 high, TCR-alpha beta low and TCR-alpha beta intermediate cells also occurred with a kinetic correlated to the reduction of CD4+CD8+ cells. Our results further showed that the percentages of V beta 8+ cells reduced 12 h post SEB injection, increased after 2 days, and decreased again thereafter. SEB thus causes both apoptotic and stimulative effects in the thymus. Apparently, the tremendous loss of double-positive cells (greater than 90% in cell number on day 4) is not simply due to the reduction of V beta 8+ cells, the possible modulatory effect of other factors or hormones which may play a role in the cell death is discussed.

Primary structure of thymosin beta 12, a new member of the beta-thymosin family isolated from perch liver.

A new polypeptide termed thymosin beta 12 has been isolated from perch liver and its primary structure elucidated. This polypeptide contains 43 amino acid residues with a molecular weight of 4822 Da. The content of thymosin beta 12 from perch liver has been determined as 43 micrograms/g of tissue. The amino-terminal end of this polypeptide is blocked by an acetyl group as deciphered by fast-atom bombardment mass spectrometric analysis. Sequence analysis reveals that thymosin beta 12 is 79% homologous to thymosin beta 4, an immunomodulator which was originally isolated from calf thymus. Thymosin beta 12 also shows 84% sequence homology to thymosin beta 11, a beta 4 analog which replaces beta 4 in two species of bony fish, oscar and rainbow trout. The evolutionary implication of such results will be discussed. The isolation of a new beta 4-related peptide from perch liver which differs from beta 11 indicates that beta-thymosin peptides are widely distributed in lower vertebrate classes.

Structure and immunological properties of thymosin beta 9 Met, a new analog of thymosin beta 4 isolated from porcine thymus.

During the course for the studies of thymosin beta 4 and prothymosin alpha from porcine thymus, a new analog of thymosin beta 4 has been identified. This peptide consists of 41 amino acid residues. The amino terminus is blocked by an acetyl group as revealed by fast atom bombardment mass spectrometric analysis. Amino acid sequence studies disclosed that this peptide is identical to bovine thymosin beta 9 except that leucine at position 6 in beta 9 is substituted by methionine. Thus, this new peptide has been termed thymosin beta 9 Met. The recoveries of beta 9 Met, beta 4, and prothymosin alpha in porcine tissues have been determined (in micrograms/g tissue) as follows: thymus (43, 85, 133); spleen (68, 203, 37); liver (10, 31, 27); heart (1.5, 10, 0); kidney (5, 51, 37); brain (0.8, 31, 5). Biologically, thymosin beta 9 Met was found to be more active than beta 4 in enhancing gamma-interferon production in cord blood lymphocytes. However, beta 4 appeared to stimulate higher amounts of interleukin 2 and tumor necrotic factor. The significance for the coexistence of two homologous peptides with similar functions in the thymus and a number of other organs is not clear, and deserves further investigation.

Depression of prothymosin alpha production in murine thymus correlates with staphylococcal enterotoxin-B-induced immunosuppression [corrected].

Prothymosin alpha [corrected] (ProT alpha) and thymosin beta 4 [corrected] (T beta 4) were isolated from murine thymus and characterized by microsequence analysis. Murine T beta 4 has an identical sequence to bovine T beta 4, whereas murine ProT alpha is highly homologous to rat Pro T alpha. Murine Pro T alpha differs from rat Pro T alpha at two positions, Glu100 and Asp108 of the rat sequence are substituted by aspartic and glutamic acid, respectively, in murine Pro T alpha. The amount of Pro T alpha in murine thymus was found to be reduced after in vivo treatment with staphylococcal enterotoxin B [corrected] (SEB), a superantigen which stimulates T cells bearing specific V beta receptors. Results from the anti-SRBC (sheep erythrocyte) plaque-forming cell assay showed that the antibody response of the spleen cells from these animals was also suppressed. On the other hand, the amount of T beta 4 was not changed significantly. Our studies suggest that the suppression of SEB on antibody response correlates with the depression of Pro T alpha production in the thymus.

Biochemical characterization of thymic hormones in thymoma tissues.

Thymic hormones induce T-cell markers and functions. These polypeptide hormones have also been shown by means of immunocytochemistry to localize in thymic epithelial cells. Employing biochemical isolation procedures, we have studied the concentration of two thymic hormones, prothymosin alpha and thymosin beta 4, in the thymus of three thymoma patients. After a brief boiling step, thymic tissue obtained from each patient was individually homogenized and centrifuged. The supernatant was then fractionated by gel filtration on Sephadex G-100 and further purified by reversed-phase high performance liquid chromatography (HPLC). Purified components were characterized by amino acid analysis and HPLC tryptic peptide mapping. Our results revealed that the extract from benign thymoma had both prothymosin alpha and thymosin beta 4, similar to normal human thymus. However, the thymus from a patient with invasive malignant thymoma contained only thymosin beta 4, but no prothymosin alpha. In the extract from an undifferentiated carcinoma, neither prothymosin alpha nor thymosin beta 4 could be detected. These results disclose the possible correlation of thymic hormones and the type and differentiation stage of thymomas. The inability of malignant thymic tumors to produce normal amounts of thymic hormones may contribute to their etiology. It is suggested that information on the thymic hormone content might add a new parameter to pathological diagnosis in thymic tumors.

A comparative study on the immunological effects of bovine and porcine thymic extracts: induction of lymphoproliferative response and enhancement of interleukin-2, gamma-interferon and tumor necrotic factor production in vitro on cord blood lymphocytes.

Forty samples of cord blood lymphocytes were isolated from 40 normal healthy full-term newborns. The initial 20 samples were used to determine the dose-response curve of three different thymic extracts (TP-1, bovine thymic extract; TG-15-I and TG-15-II, both porcine thymic extracts) and one of renal origin (KG-1) as a control of non-lymphoid organ extract, by measuring the E-rosette T cells. Results showed that E-rosette T cells increased significantly when the thymic extract concentration was increased to 12.5 micrograms/ml. However, there was no statistical difference between TP-1, TG-15-I and TG-15-II in the increase of E-rosette-forming cells. The remaining 20 samples were preincubated with 0, 12.5, 25 or 50 micrograms/ml of thymic extracts. It was observed that the lymphoproliferation, interleukin-2 (IL-2), gamma-interferon (IFN-gamma) and tumor necrotic factor (TNF) production were all significantly increased after thymic extract treatment. No statistical difference between these three thymic preparations in the stimulation of lymphoproliferative response was found. However, among the three thymic extracts, TP-1 appears to induce the highest amounts of IL-2, IFN-gamma and TNF. Of the TG-15-I and TG-15-II, the former stimulates higher IL-2 production whereas the latter enhances IFN-gamma and TNF production. The different immunostimulating effects and potencies that these three thymic extracts showed may reflect not only the species difference but also the difference in preparation procedures. Different components in these thymic extracts may be responsible for different biological activities. Results from these comparative studies may provide useful information in future clinical trials for the treatment of the primary immunodeficiency diseases according to their pathogenesis and may also indicate a possible beneficial effect of the combination of chemotherapy and thymic extracts.

Isolation and structural studies of porcine, ovine and murine thymosin beta 4 by high-performance liquid chromatography.

Rapid high-performance liquid chromatographic (HPLC) procedures have been used to isolate and characterize thymosin beta 4 from different species. Crude extracts termed thymosin fraction 5A were prepared from porcine and ovine thymus glands as well as murine spleen. Each fraction 5A preparation was then fractionated by HPLC on a muBondapak C18 reversed-phase column. Porcine and ovine thymus fraction 5A, and murine spleen 5A, each yields a predominant peak at a retention time similar to that of bovine thymosin beta 4. Amino acid analysis as well as HPLC tryptic peptide mapping of these peaks indicate that they have homologous sequences to bovine thymosin beta 4. Chromatographic analysis of fresh murine thymus and spleen tissues also revealed protein peaks at the position of bovine beta 4, suggesting that thymosin beta 4 is the native protein present in the animal tissues.

Immunochemical studies on thymosin: radioimmunoassay of thymosin beta 4.

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.

Thymosins: structure, function and therapeutic applications.

Using a partially purified preparation, thymosin fraction 5, we have documented that thymosin can correct many of the immunological deficiencies resulting from the lack of thymosin function in animal models and in humans. Ongoing studies indicate that there is a family of biologically active peptides within fraction 5 that act on T-cell subpopulations to maintain normal immunological reactivity. Several of these peptides have been purified to homogeneity. Two peptides, thymosin alpha 1 and beta 4, have been sequenced and chemically synthesized. Thymosin fraction 5 has been used in most clinical trials reported to date, including children with immunodeficiency disease and patients with autoimmune diseases and cancer. Most recently, the National Cancer Institute has initiated a number of Phase I and Phase II clinical trials with thymosin fraction 5 and synthetic alpha 1 as part of a new Biological Response Modifier Program. Preliminary results from two of these studies look encouraging.

Restorative effects of thymosin polypeptides on purified protein derivative--dependent migration inhibition factor production by the peripheral blood lymphocytes of adult thymectomized guinea pigs.

Thymosin fraction 5 restored the capacity of peripheral blood lymphocytes (PBL) from thymectomized adult guinea pigs sensitized with purified protein derivative (PPD) to produce migration inhibition factor (MIF) as measured in an agarose droplet microassay with peritoneal exudate cells. PBL from sham-thymectomized and normal PPD-sensitized guinea pigs did not lose their capacity to make MIF in response to PPD and were unaffected by thymosin polypeptides. Splenic and lymph node lymphocytes of thymectomized guinea pigs retained their antigen responsivity , suggesting that thymectomy did not affect fully matured, localized memory cells, but caused an accumulation in the circulation of partially matured, antigen-sensitive lymphocytes that could release MIF only in the presence of thymic factors. Various thymosin polypeptides were tested in this system and only thymosin alpha 1, an acidic polypeptide of 28 amino acid residues, showed consistent activity. This polypeptide, which has a molecular weight of 3,108, did not inhibit macrophage migration by itself, but 10-1,000 ng/ml was able to completely restore the MIF-producing capability of PBL from thymectomized guinea pigs. A standardized method for pooling and cryopreserving guinea pig PBL was developed, allowing multiple assays to be performed with a single pool of cells. The specificity of this bioassay system has made it a useful tool in evaluating the thymosin alpha 1 activity present in various thymosin preparations, and in validating biological activity of thymosin alpha 1 preparations made by synthetic or recombinant DNA techniques.

Isolation of thymosin alpha 1 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.

Solid-phase synthesis of thymosin beta 4: chemical and biological characterization of the synthetic peptide.

The chemical synthesis of thymosin beta 4 using a solid-phase procedure has been accomplished. The synthetic product was found to be homogeneous on paper electrophoresis at pH 6.5, high-performance liquid chromatography on a reversed-phase column, and isoelectric focusing using polyacrylamide gels. The synthetic material was also shown to be identical with the natural thymosin beta 4 by tryptic peptide mapping, amino acid compositional analyses, and polyacrylamide gel isoelectric focusing. Biologically, synthetic thymosin beta 4 was found to be as active as the natural compound in a terminal deoxynucleotidyltransferase induction assay and in a macrophage migration inhibition assay. The proposed structure of the peptide hormone was thus confirmed by a chemical synthesis.

Chemical characterization of thymosin beta 4.

As part of our ongoing investigations on the endocrine thymus, we have isolated and purified to homogeneity a hormone-like peptide which we have termed thymosin beta 4. Thymosin beta 4 has Mr = 4982 and an isoelectric point of 5.1. The complete amino acid sequence of this polypeptide has been established by automated Edman degradation as well as by manual sequence analysis. Thymosin beta 4 is composed of 43 amino acid residues with acetylserine at the NH2 terminus. This molecule induces expression of terminal deoxynucleotidyl transferase in transferase-negative murine thymocytes in vivo and in vitro. It also exhibits ability to inhibit the migration of macrophages. Comparison of the sequence of thymosin beta 4 to other thymic hormones or other published protein sequences does not reveal any statistically significant relationship. Two helical regions were identified in the structure using data for prediction of protein conformation. It is proposed that thymosin beta 4 is one of the biologically active peptides present in thymosin fractions 5 and 5A which participate in the regulation, differentiation, and function of thymus-derived lymphocytes and may also act directly or indirectly on macrophages and perhaps other cells involved in cell-mediated immunity.

Modulation of terminal deoxynucleotidyl transferase activity by thymosin.

Thymosin fraction 5, a family of acidic polypeptides isolated from bovine thymus, contains several hormonal-like factors which have been shown to influence the maturation, differentiation and functions of T-cells. Some of these peptides have been chemically defined. Two of them, thymosin alpha 1 (M.W. 3108) and thymosin beta 4 (M.W. 4982) have been sequenced. In murine systems, terminal deoxynucleotidyl transferase (TdT) has been shown to be T-cell specific and to be present primarily in the cortisone sensitive immature T-cell populations. The daily injection of thymosin fraction 5 and two of its components, thymosin beta 3 and beta 4, significantly increases TdT activity in immune suppressed mice as compared to control groups. This study indicates that thymosin can act on prothymocytes and influence the early stages of T-cell differentiation. In an in vivo system, thymosin fraction 5 and the purified peptide, thymosin alpha 1, have high activities in decreasing TdT in normal murine thymocytes after a 22-h incubation. This effect suggests that thymosin can also act on thymocytes and regulate the later biochemical processes during T-cell differentiation.