Paul H. Naylor

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.

Thymosin α1 and thymosin β4 in serum: Comparison of normal, cord, homosexual and AIDS serum

Thymosin alpha 1 and thymosin beta 4 were first isolated from thymosin fr. 5 and have demonstrated biological activities on the immune system. They are chemically distinct and differ in their immunological activity profiles. The levels of thymosin alpha 1 and thymosin beta 4 were assessed by radioimmunoassay in the same serum samples. Normal thymosin alpha 1 levels were 670 +/- 163 pg/ml for males and 652 +/- 162 pg/ml for females. Normal thymosin beta 4 levels were 974 +/- 400 ng/ml for males and 889 +/- 345 ng/ml for females. No correlation between the levels of the peptides in serum from normal donors was observed. Although many samples of serum from neonates (cord blood), homosexuals and AIDS patients had elevated levels of one or both peptides, no correlation between the two peptides was found. Of potential significance is the observation that while thymosin alpha 1 and beta 4 are elevated in many individuals with AIDS (57 and 48% respectively), the individuals with AIDS related immune dysfunctions had predominantly elevated thymosin alpha 1 (54 vs 15%). These studies suggest that serum levels of the two peptides are modulated separately and that both are of potential value in defining the risk of individuals for developing AIDS.

Specific antibody synthesis in vitro. II. Age-associated thymosin enhancement of antitetanus antibody synthesis

A decline in T cell function accounts for many of the observed age-related deficient immune responses. Specific antibody response to many antigens requires T cell cooperation, and deficient antibody response to such antigens has been demonstrated with aging. In an effort to assess the potential reconstitutive capacity of Thymosin Fraction 5, in vitro antitetanus antibody production was measured in tetanus toxoid booster-immunized young and old volunteers. 22 young and 12 old volunteers were immunized with tetanus toxoid and plasma antitetanus antibody and in vitro lymphocyte production of antitetanus antibody was measured. Plasma antitetanus antibody response was significantly greater in the young. In vitro antitetanus antibody synthesis was negligible prior to immunization and peaked in cultures established 1 week after immunization from both young and old. When Thymosin Fraction 5 was added to the cultures, however, there was a dose-related enhancement of antibody synthesis in 7 of 10 from the group of elderly volunteers, but only 3 of 12 from the younger group. Our data indicate that specific antibody response is deficient in the elderly, but can be enhanced in vitro by thymosin. A future clinical trial of thymosin as an adjuvant to active immunization for the elderly is warranted.

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.

Human T cell leukemia virus type III antibody, lymphadenopathy, and acquired immune deficiency syndrome in hemophiliac subjects. Results of a prospective study

A cohort of 63 hemophiliac subjects was followed for clinical and immunologic abnormalities related to the acquired immune deficiency syndrome (AIDS). When evaluated in early 1984, antibody to human T cell leukemia virus type III (HTLV-III) was detected in the serum of 59 percent (24 of 41) of factor VIII or IX concentrate recipients, but in none (0 of six) of the cryoprecipitate/fresh frozen plasma recipients. HTLV-III-seropositive hemophiliac subjects, on average, had been exposed to twice as much concentrate during the previous year as seronegative hemophiliac subjects. The seropositive group had a significantly lower mean helper/suppressor T cell ratio and absolute helper T cell level than the seronegative group. By early 1984, 13 hemophiliac subjects in the study population had lymphadenopathy and one had AIDS. Antibody to HTLV-III was detected in the serum of 13 of these 14 hemophiliac subjects with overt clinical disease. The prevalence of lymphadenopathy or AIDS among HTLV-III-seropositive hemophiliac subjects was 54 percent (13 of 24). It is concluded that HTLV-III antibody occurs with high frequency in hemophiliac subjects, and is related to the amount of factor VIII or IX concentrate infused. Over half of HTLV-III-seropositive hemophiliac subjects in this population had overt clinical disease with either lymphadenopathy or AIDS.

Antibody to Human T-Cell Leukemia Virus Membrane Antigens, Beta2-Microglobulin Levels, and Thymosin Alpha1 Levels in Hemophiliacs and Their Spouses

Recently, antibodies to human T-cell leukemia virus membrane antigens (HTLV-MA) and elevated levels of beta 2-microglobulin and thymosin alpha 1 have been found with high frequency in patients with the acquired immunodeficiency syndrome. Prospective studies of asymptomatic persons at high risk for this syndrome will ascertain whether any of these findings is a predictive marker for the disease. In this study, antibodies to HTLV-MA, beta 2-microglobulin levels, and thymosin alpha 1 levels were determined for a group of asymptomatic adult hemophiliacs and their wives. Five of thirty-nine hemophiliacs had HTLV-MA antibody, compared with none of 21 wives tested. The mean beta 2-microglobulin level for hemophiliacs was significantly higher than the control value (p less than 0.001), whereas the wives had a normal mean value. The mean thymosin alpha 1 values were normal for hemophiliacs and their wives; however, 3 of 22 hemophiliacs and 1 of 16 wives had abnormally high levels. Whether any of these abnormalities correlate with subsequent development of the acquired immunodeficiency syndrome will be ascertained by longitudinal follow-up of this population.

Thymosin: Cyclic nucleotides and T cell differentiation

Abstract This review summarizes the evidence suggesting a role for second messengers in the thymus dependent differentiation of lymphocytes. Special emphasis will be placed on the potential for such studies to indicate differences and similarities not only between thymosin and other thumus factors but between the various peptides which are present in thymosin fraction 5.

Immunopharmacology of Thymosin α1 and Cytokine Synergy

Abstract:  Thymosin α1 (Tα1) is a 28 amino acid biologically active protein cleaved from positions 2–29 of a precursor protein, prothymosin α. Since its discovery, Tα1 has been administered to animals and humans in a wide variety of settings and its pharmacologic effects are to enhance cellular immunity. Tα1 administration is highly effective in settings where irradiation, chemotherapy, tumor burden, or immune senescence have caused a reduction of T cell number and/or function. Recent in vitro studies, including the one reported here, suggest that Tα1 may act via pathways commonly used by various cytokines. This raises the possibility that Tα1 and cytokines may have synergistic activity through potentiation of cytokine activity by Tα1. Improved control of tumor growth when tumor‐bearing mice were treated with Tα1 and high doses of IL‐2 has been previously reported. We extended those studies with the Lewis lung carcinoma mouse model using IRX‐2, a natural well‐defined biologic containing multiple cytokines, in combination with Tα1 (IRX‐3). Although IRX‐2 was effective alone (using doses that contain significantly less IL‐2 than in most typical studies), adding Tα1 led to significant improvement in survival of the tumor‐bearing mice. Based on these observations, the immunopharmacology of Tα1 predicts an important clinical role for Tα1 in the restoration of cellular immune activity when used in combination with cytokines. Patients who experience immune suppression due to the presence of tumor, irradiation, and/or chemotherapy or aging of the host would most benefit from this treatment combination.

MicroELISA method for measurement of human serum thymosin α1

Abstract A microELISA for the estimation of human serum thymosin α1 is described. In this assay, antibody to thymosin α1 is pre-incubated with the standard or serum at 4°C. Unbound antibody in the liquid-phase then binds with solid-phase thymosin α1. The method is sufficiently sensitive for measuring serum levels of thymosin α1 and highly reproducible. The serum levels measured with the microELISA are comparable to serum levels of thymosin α1 determined by the previously described radioimmunoassay for thymosin α1.

Circulating thymulin and thymosin-α1 activity in pediatric acquired immune deficiency syndrome: In vivo and in vitro studies

Twenty-five children with acquired immune deficiency syndrome (AIDS) or AIDS-related complex had a characteristic pattern of T cell deficiency. Abnormally low plasma thymulin levels preceded the development of peripheral blood T cell abnormalities. In contrast to patients with congenital T cell deficiencies, our patients had elevated serum levels of thymosin-alpha 1. Treatment with thymosin fraction 5 in three children with AIDS resulted in only transient clinical and immunologic improvement.