Thymosin Fraction Research Articles

Smoking Abstinence and Small Cell Lung Cancer Survival

The prognostic implications of cigarette smoking were investigated in 112 patients with small cell lung cancer. Twenty had stopped smoking permanently before diagnosis (NS-Prior), 35 had stopped at diagnosis (NS-Dx), and 57 patients continued smoking (S). Therapies included chemotherapy alone or with radiation therapy, with or without thymosin fraction V. The survival difference among the three groups was statistically significant. The NS-Prior patients had the best survival, followed by NS-Dx patients and finally S patients. No S patient has survived, disease free, more than 96 weeks, while three NS-Prior and three NS-Dx patients are disease free 103 to 220 weeks after start of treatment. Thymosin, 60 mg/sq m, yielded survival benefits for the S group only. Continuation of smoking during the treatment of small cell lung cancer was associated with a poor prognosis, while discontinuation of smoking, even at diagnosis, may have beneficial effects on survival.

Lymphocyte cytotoxicity in human liver disease using rat hepatocyte monolayer cultures

Isolated rat hepatocytes were used to determine the cytotoxicity of peripheral blood mononuclear cells (PBM) from patients with alcoholic liver disease (ALD) and chronic active hepatitis (CAH). The specificity of the cytotoxic effect on liver cells was monitored using rat kidney cells. PBM from patients with CAH and ALD showed increased spontaneous cell-mediated cytolysis (SCMC) for hepatocytes. The SCMC against kidney cells was comparable for the patient groups and controls. Incubation of liver cells with antibody to human liver-specific protein significantly increased the cytotoxic activity of control PBM but did not block cytotoxicity by PBM of patients with CAH. Incubation of PBM from untreated CAH patients with thymosin fraction 5, a polypeptide extract of the thymus gland, significantly lowered cytotoxicity. Our findings suggest that rat liver cells provide a model for studies of cell-mediated immunity in human liver disease and that thymosin fraction 5 decreases the cytotoxic activity of sensitized lymphocytes in CAH.

Thymosin-dependent T-lymphocyte response in inflammatory bowel disease

Peripheral blood “total” and “avid” thymus-dependent (T) lymphocytes were enumerated in 45 patients with Crohn's disease (CD) and in 23 patients with ulcerative colitis (UC) by using the spontaneous rosette technique (ER). The in vitro effect of thymosin fraction 5, a polypeptide extract of the thymus gland, on avid ER formation was also determined in these patients. The proportion and number of “total” ER were lower in patients with CD (P < 0.02), but not with UC, when compared with controls. More impressive differences were observed when “avid” ER were determined in patients with CD (P < 0.001) and UC (P < 0.05). Incubation with thymosin resulted in a significant increase in “avid” ER in patients with CD and UC, with no such effect observed in the controls. These results indicate that the determination of “avid” rather than “total” ER provides a more sensitive method for detecting alterations in T-cell immune competence. In addition, it is suggested that there is an increased number of circulating T-lymphocytes in CD and UC capable of responding to exogenous thymic factors. This may indicate the presence of a thymosin-responsive immunodeficiency state in these diseases.

Thymosin therapy in the DiGeorge syndrome.

Reconstruction of the T-cell immune defect in patients with the DiGeorge syndrome has been accomplished in the past by fetal thymus transplantation. Because of the risk of fatal graft-versus-host reaction with fetal thymus transplantation in patients with abnormal T-cell immunity, we have examined the effects of a thymus tissue extract, thymosin fraction 5, on the in vitro and in vivo immune function in children with the DiGeorge syndrome. T-cell numbers were increased with thymosin F5 in vitro in three of five patients. T-cell number and function was improved in three of four patients treated with thymosin F5 in vivo. Spontaneous improvement in the immune function of these patients cannot be excluded. These results suggest, however, that further trials with thymosin F5 therapy may be indicated in patients with the DiGeorge syndrome.

Effect of thymosin on T-lymphocyte rosette formation in children with kwashiorkor

Thymosin fraction 5 causes a significant increase in vitro in the percentage of E-rosettes (T-cells) of children with kwashiorkor. It is speculated that thymosin fraction 5 may play an important role in reconstituting host immunity in children with malnutrition.

Thymosin-induced T cell marker expression and enhanced mitogen responsiveness in allogeneic marrow transplant recipients.

Thymosin fraction V, prepared from calf thymus glands was incubated with lymphoid cells from allogeneic bone marrow transplant recipients to determine the effects in vitro of thymosin on the expression of a T cell surface marker and on T mitogen-induced proliferative responses. Between 2 and 7 weeks after transplant, thymosin, after a brief coincubation, induced additional patient lymphoid cells to express the receptor for sheep erythrocytes. Between 1 and 8 months after transplant, thymosin, after an overnight preincubation, induced enhanced proliferative responses to phytohemagglutinin, concanavalin A, and occasionally pokeweed mitogen. These data suggest that marrow transplant patients had circulating lymphoid cells that were sensitive to thymosin, Thymosin induced the expression of a T cell receptor and enhanced the performance of a T cell functional response. Thymosin appeared to drive the maturation sequence of marrow transplant patient peripheral T cells. These studies were performed at a time when the allogeneic marrow transplant recipients were markedly immunodeficient and susceptible to opportunistic infections. These results suggest that thymosin fraction V may be useful to stimulate the immune system in vivo in patients after allogeneic marrow transplantation.

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.

THYMOSIN MODULATION OF SUPPRESSOR FUNCTION IN MICE AND MAN1

The effect of thymosin on suppressor-cell function was evaluated in vivo in a murine tumor system and in vitro on human lymphocytes. In mice, the Lewis tumor system was used. We showed that splenocytes from tumor-bearing animals were able to enhance tumor growth in a syngeneic system. This enhancement was similar when thymocytes from tumor-bearing animals were used and disappeared after anti-Thy 1-2 antiserum treatment, suggesting a T-dependence. Treatment of the tumor-growth-enhancing lymphocytes with corticosteroids or irradiation caused this effect to disappear completely suggesting that the tumor-growth-enhancing T-lymphocytes were suppressor T-cells. Furthermore thymosin (fraction 5)-treated, tumor-growth-enhancing T-lymphocytes were not able to enhance tumor growth and even significantly decreased it. In the human system we showed that Con A-stimulated lymphocytes were able to suppress the response of normal lymphocytes to PHA, PWM, and Con A, and in MLC. This effect was significantly blocked in presence of thymosin fraction 5.

The prevalence of autoantibodies in T-cell, B-cell, and phagocytic immunodeficiency disorders

Patients with a spectrum of T-cell, B-cell, and phagocytic disorders were studied to correlate the specific immunodeficiency associated with autoantibody and autoimmune disease. Disorders investigated included X-linked hypo-γ-globulinemia, common variable onset hypo-γ-globulinemia, selective IgA deficiency, cellular immunodeficiency with abnormal immunoglobulin synthesis, Wiskott-Aldrich syndrome, ataxia-telangiectasia, chronic mucocutaneous candidiasis, severe combined immunodeficiency, chronic granulomatous disease, and female carriers of chronic granulomatous disease. Autoantibody was detected in all groups except X-linked hypo-γ-globulinemia. Clinical autoimmune disease was observed in patients with X-linked hypo-γ-globulinemia, common variable onset hypo-γ-globulinemia, selective IgA deficiency, Wiskott-Aldrich syndrome, chronic mucocutaneous candidiasis, chronic granulomatous disease, and female carriers of chronic granulomatous disease. No autoantibodies were found in only one immunodeficiency disorder, X-linked hypo-γ-globulinemia. Partial B-cell dysfunction, T-cell dysfunction, and phagocytic dysfunction predispose to autoantibody formation. Clinical autoimmune disorders were found in all types of immunodeficiency except severe combined immunodeficiency. Alternatively, patients with partial deficiencies of both B- and T-cell immunity may have regulator cell defects of helper versus suppressor cell populations which may result in enhanced autoantibody formation. It is of interest that two of the patients with cellular immunodeficiency and abnormal immunoglobulin synthesis who were initially negative for autoantibodies, developed positive autoantibodies after partial reconstruction of T-cell function following treatment with bovine thymosin fraction 5 and fetal thymus transplantation, suggesting that under certain circumstances, enhanced T-cell function may result in autoantibody formation.

Effect of calcium on the cyclic GMP elevation induced by thymosin fraction 5

Thymosin fraction 5 induces an increase in cyclic GMP but not cAMP in murine thymocytes. Calcium (0.6 mM) is necessary for an optimal response in both phosphate buffered saline and hepes-buffered RPMI 1640 media. The calcium dependence of the cGMP response was most pronounced in a minimal salts medium (PBS) and higher concentrations (greater than 0.8 mM) caused a lessening of the cGMP elevation induced by thymosin. Basal cGMP levels of thymus and spleen lymphocytes vary with increasing concentrations of calcium (0–1 mM) and to a lesser extent, the levels of cAMP also are increased. Calcium uptake was measured both at mitogenic levels of Con A and at thymosin concentrations which were similar to those necessary for the increase in cGMP. The results suggest that calcium and cGMP play an important role in T cell differentiation under the influence of thymosin.

Immunosuppression and reconstitution with thymosin after radiation therapy

Since 1976, 102 patients with advanced squamous cell carcinoma, (82, head and neck; 20, esophagus), have been evaluated before receiving irradiation and thymosin fraction 5 therapy at the University of California, San Francisco (UCSF). Thymosin fraction 5 is a mixture of at least 20 polypeptides which has been shown to have immune enhancing capabilities in primary immunodeficiency disease and various malignancies. Immunity prior to treatment (measured by total lymphocyte count, E and EAC rosettes, lymphocyte stimulation with phytohemagglutinin (PHA) and in mixed leukocyte culture (MLC) with allogeneic cells and quantitative serum immunoglobulins) was comparable and normal in the 80 control patients and the 22 thymosin treated patients. Postirradiation significant depression ( p < 0.01) was demonstrated in cellular immunity in both groups of patients with decreased T and B cell numbers and depressed PHA and MLC stimulation. To date there have been an equal number of recurrences and deaths in both groups of patients.

Effect of thymosin on PHA and Con A responses by fetal lamb lymphocytes

The effects of thymosin fraction 5 on the mitogen responses of fetal lamb lymphocytes in serum-free cultures were studied. The cells from liver, thymus, spleen, and bone marrow of 12 fetal lambs of four different ewes at 48–98 days of gestation (total gestation is 150 days) were used. Cells from the liver, bone marrow, and spleen were separated by Ficoll-Isopaque density-gradient centrifugation. At 48 to 68 days of gestation, fetal liver cells had only a slight capacity to respond to PHA and Con A mitogens; however, these responses could be induced by thymosin. Later on, when the fetal lymphocytes including those from liver, thymus, spleen, and bone marrow started to respond to mitogens (from Day 78 onward), the effect of thymosin became negligible.

Thymosin fraction V and intensive combination chemotherapy. Prolonging the survival of patients with small-cell lung cancer

Patients with small-cell bronchogenic carcinoma who received intensive remission-induction chemotherapy randomly received either thymosin fraction V, 60 mg/sq m or 20 mg/sq m twice weekly, or no thymosin treatment during the initial six weeks of chemotherapy. Chemotherapy was then continued for two years. Thymosin administration did not increase the complete response rate. Patients receiving thymosin, 60 mg/sq m, had significantly prolonged survival times relative to the other treatment groups. This benefit was due to prolonged relapse-free survival in complete responders to treatment. The mechanism by which thymosin increased survival duration is unclear but may relate to restoration of immune deficits due to disease or treatment.

Thymosin Fraction V and Intensive Combination Chemotherapy

Patients with small-cell bronchogenic carcinoma who received intensive remission-induction chemotherapy randomly received either thymosin fraction V, 60 mg/sq m or 20 mg/sq m twice weekly, or no thymosin treatment during the initial six weeks of chemotherapy. Chemotherapy was then continued for two years. Thymosin administration did not increase the complete response rate. Patients receiving thymosin, 60 mg/sq m, had significantly prolonged survival times relative to the other treatment groups. This benefit was due to prolonged relapse-free survival in complete responders to treatment. The mechanism by which thymosin increased survival duration is unclear but may relate to restoration of immune deficits due to disease or treatment.

Failure of NZB Spleen to Respond to Prethymic Bone Marrow Suppressor Cells

Mouse bone marrow contains theta-negative lymphocytes that can suppress an in vitro plaque response by spleen cells primed in vivo with burro red blood cells (BRBC). These bone marrow cells are radiosensitive and can be induced with thymosin fraction 5 or alpha 1 thymic peptides to express the theta antigen. Enrichment for these suppressor pre-T lymphocytes can be achieved by a one-step density centrifugation, macrophage depletion, or a combination of both procedures. NZB mice, which spontaneously develop an autoimmune disorder, have a suppressor abnormality revealed by this assay system. Upon analysis, they have normal BM pre-T suppressor cells but their spleen cells are refractory to the BM suppressor signal. NZB BM suppressor cells inhibit the response by DBA/2 spleen cells, but DBA/2 BM suppressor cells do not inhibit NZB spleen. This resistance to suppression is a property of the B cell fraction recovered from NZB spleen.

Cyclic nucleotide changes in murine lymphocytes following thymosin incubation in vitro

Thymosin fraction 5 and a more purified acidic fraction of thymosin at a 100-fold lower concentration elevated cGMP levels, but not cAMP levels, in murine thymocytes. When both thymus and spleen lymphocytes were fractionated via a BSA gradient procedure, both cAMP and cGMP basal values varied depending on the density of the subpopulations. Thymosin Fr5 did not elevate cAMP in any thymus subpopulations of lymphocytes obtained from the BSA density gradients. The cGMP elevation due to thymosin Fr5 in thymocytes was maximal in the most buoyant thymocytes, and no elevation of cGMP was detected in nude mouse spleen lymphocytes. These results suggest that the cGMP elevation may be an early event in the thymosin-mediated differentiation of a more mature subpopulation of thymocytes. They also suggest that utilization of subpopulations may be necessary for the complete determination of the effects of agents on cyclic nucleotide values of lymphocytes.

In vitro thymosin effect on T lymphocytes in alcoholic liver disease

The in vitro effect of thymosin fraction 5, a thymic gland extract, on thymus-dependent lymphocytes was studied in 12 patients with alcoholic hepatitis (AH) and in 18 patients with compensated alcoholic cirrhosis (CAC). The baseline number and proportion of spontaneous rosette-forming T lymphocytes were significantly reduced in AH as compared to the controls. No such decrease was seen in CAC. When the lymphocytes of patients with AH were incubated in the presence of thymosin fraction 5, the subsequent number and proportion of rosette-forming T lymphocytes showed a significant increase. There was no significant reponse to thymosin in CAC or in control subjects. There appears to be a subset of immature T lymphocytes in patients with AH that is responsive to exogenous thymic factors with subsequent activation of the capacity to form spontaneous rosettes.

The chemistry and biology of thymosin. I. Isolation, characterization, and biological activities of thymosin alpha1 and polypeptide beta1 from calf thymus.

A partially purified extract from thymus tissue termed thymosin Fraction 5 has been shown to reconstitute immunological deficiencies resulting from the lack of thymic function in several animal models, as well as humans with primary and secondary immunodeficiency diseases. Thymosin Fraction 5 consists of a family of polypeptides with molecular weights ranging from 1,000 to 15,000. Several of these polypeptides contribute individually to the biological activity of the parent compound. Two polypeptide components of thymosin Fraction 5, termed thymosin alpha1 and polypeptide beta1, have been characterized chemically and biologically. Thymosin alpha1 is a highly acidic molecule composed of 28 amino acid residues. This polypeptide has potent biological activity and has been found to be 10 to 1,000 times as active as thymosin Fraction 5 in one in vivo and several in vitro bioassay systems designed to measure differentiation and function of thymus-dependent lymphocytes (T cells). Polypeptide beta1, in contrast, is inactive in our bioassay systems, suggesting that it is not involved in thymic hormone action. Sequence analysis and homology studies have indicated that polypeptide beta1, although present in Fraction 5, does not contribute to the biological activity of thymosin Fraction 5.

The chemistry and biology of thymosin. II. Amino acid sequence analysis of thymosin alpha1 and polypeptide beta1.

The amino acid sequences of two polypeptide components of thymosin Fraction 5 termed thymosin alpha1 and polypeptide beta1 have been established. The sequences were determined by automatic Edman degradation of the intact molecules as well as by manual sequence analysis of the enzymatic cleavage products. Thymosin alpha1, an immunologically active polypeptide, is highly acidic with an isoelectric point of 4.2. This molecule is composed of 28 amino acid residues with acetylserine as the NH2 terminus. A chemically synthesized molecule of thymosin alpha1 has been found to be as active as the natural molecule in our bioassay systems. Polypeptide beta1 is a molecule consisting of 74 amino acid residues and has an isoelectric point of 6.7. This peptide is not biologically active in our assay systems, suggesting that it is not involved in thymic hormone action. The sequence of beta1 was found to be identical with ubiquitin and a portion of protein A24, a nuclear chromosomal protein. The relationships among these proteins are discussed.

Effect of thymosin on the mitogenic responses of guinea pig thymocytes with different maturation stages

Unfractionated guinea pig thymocytes were preincubated for two hours with 14 different concentrations of thymosin fraction 5 and thereafter the thymocytes were stimulated with mitogens. Three concentrations (0.05, 10 and 200 μg/ml) of thymosin significantly increased the PHA response of thymocytes; one concentration (0.05 μg/ml) significantly increased the Con A response. The results were not due to thymosin-induced changes in the PHA or Con A dose-response curves. Thymosin fraction 5 was also tested on two subpopulations of thymocytes at different stages of maturation. Thymosin did not increase PHA or Con A responses in the immature subpopulation. In contrast, it induced a significant inhibition of proliferation in these cells. Thymosin concentrations of 0.05 and 10 μg/ml significantly increased the PHA and Con A responses of the more mature subpopulation of thymocytes. The results support the concept that thymosin fraction 5 at concentrations of 0.05 and 10 μg/ml convert thymocytes into more mature PHA and Con A reactive cells. Higher concentration of thymosin (200 μg/ml) probably induces maturation of thymocytes to PHA reactive cells. These findings suggest that various concentrations of thymosin act to mature thymocytes at different stages of maturation; higher concentrations of thymosin are required to affect immature thymocytes than are required to affect more mature cells.