Thymosin Fraction Research Articles

Neurochemical study of the mechanism of action of thymus peptides in emotional stress

A comparative study of the effect of the thymus hormone thymulin and thymus peptides (thymosin fraction 5) on the content of monoamines and their metabolites in the frontal cortex, striatum, adjacent nucleus, hypothalamus, hippocampus of the brain of Wistar rats, by high performance liquid chromatography, was performed. The hormone and peptides of the thymus were found to reduce emotional stress during functional impairment ofthe avoidance reaction and improved adaptation under stressful conditions in rats, which indicates the anti-stress effect of thymus hormones. The positive effect of the hormone and peptides of the thymus were manifested in a change in the balance of serotonin and norepinephrine in favor of the former in the hypothalamus and frontal cortex.

ABNORMALITY IN LEARNING ON THE MODEL OF PARKINSON-LIKE SYNDROME: THE CORRECTABILITY BY THYMUS PEPTIDES

In recent times the question of the bidirectional interactions of the neuroendocrine and immune systems is studied quite actively, its mechanisms offer the opportunity to allow for optimization the therapy of several diseases, including Parkinson’s disease. The purpose of this work is to study the influence of peptide drugs derived from the body’s immune system, the thymus, for the avoidance test conditioning, both in health and in the model of Parkinson-like syndrome. The work was performed on 80 male Wistar rats weighing 300-350 g. The injection of thymosin fraction 5 and thymalin to animals corrects clinical traits and behavioral dysfunctions caused by the singular injection of the neurotoxin. It also facilitates conditioned active evasion response in rats, which is especially noticeable at the early stages of memory footprint formation.

Thymosin fraction 5 re-evaluated after 35 years by high-resolution mass spectrometry

ABSTRACTObjectives: We reevaluated a lyophilized sample of thymosin fraction 5, stored for 37 years at room temperature, by high-resolution mass spectrometry in terms of stability and yet uncharacterized polypeptides that could be biological important substances.Methods: A top–down proteomic platform based on high-performance liquid chromatography (HPLC) coupled to high-resolution LTQ-Orbitrap mass spectrometry (MS) was applied to molecular characterization of polypeptides present in thymosin fraction 5.Results: We detected more than 100 monoisotopic masses corresponding to thymosin β4 and truncated forms of ubiquitin, prothymosin α, thymosin β4, and thymosin β9. Additionally, we discovered a new polypeptide present in thymosin fraction 5 and identified it as intact SH3 domain-binding glutamic acid-rich-like protein 3.Conclusion: In spite of the well-known proteolytic processes inherent to the preparation of thymosin fraction 5, still uncharacterized polypeptides as well as truncated forms of already well-known thymosins are present in fraction 5 after long-term storage. Therefore, continuing characterization of thymosin fraction 5 is even nowadays highly promising.

Prothymosin Alpha and Immune Responses: Are We Close to Potential Clinical Applications?

The thymus gland produces soluble molecules, which mediate significant immune functions. The first biologically active thymic extract was thymosin fraction V, the fractionation of which led to the isolation of a series of immunoactive polypeptides, including prothymosin alpha (proTα).ProTα displays a dual role, intracellularly as a survival and proliferation mediator and extracellularly as a biological response modifier. Accordingly, inside the cell, proTα is implicated in crucial intracellular circuits and may serve as a surrogate tumor biomarker, but when found outside the cell, it could be used as a therapeutic agent for treating immune system deficiencies. In fact, proTα possesses pleiotropic adjuvant activity and a series of immunomodulatory effects (eg, anticancer, antiviral, neuroprotective, cardioprotective). Moreover, several reports suggest that the variable activity of proTα might be exerted through different parts of the molecule. We first reported that the main immunoactive region of proTα is the carboxy-terminal decapeptide proTα(100–109). In conjunction with data from others, we also revealed that proTα and proTα(100–109) signal through Toll-like receptor 4. Although their precise molecular mechanism of action is yet not fully elucidated, proTα and proTα(100–109) are viewed as candidate adjuvants for cancer immunotherapy.Here, we present a historical overview on the discovery and isolation of thymosins with emphasis on proTα and data on some immune-related new activities of the polypeptide and smaller immunostimulatory peptides thereof. Finally, we propose a compiled scenario on proTα’s mode of action, which could eventually contribute to its clinical application.

The impact of immunoactive drugs on passive avoidance response

The objective of this project was to explore the influence of immunoactive drugs (tactivin, thymulin, and thymosin fraction 5) on the development of the passive avoidance conditioned reflex. Two types of passive avoidance boxes were used--a regular two-chamber box and a modified three-chamber box, comprising a dark chamber in which rats were exposed to electrical shock, a safe dark chamber, and a light chamber in the center. The project has established that the memory trace persists longer under the influence of the immunoactive drugs in both models, which is consistent with the reference nootropic piracetam test results. Notably, the immunoactive drugs' mnemotropic effect was more pronounced in the modified three-chamber box than in the standard two-chamber box. Using the modified box helped to establish the influence of tactivin, thymulin, and thymosin fraction 5 on the spatial memory component. Immunotropic preparations from thymus caused the animals to select the safe chamber 24 hours later and in subsequent tests. The project's results indicate that the drugs tested do possess mnemotropic properties, so their range of clinical use can be broadened.

Prothymosin alpha: a ubiquitous polypeptide with potential use in cancer diagnosis and therapy.

The thymus is a central lymphoid organ with crucial role in generating T cells and maintaining homeostasis of the immune system. More than 30 peptides, initially referred to as "thymic hormones," are produced by this gland. Although the majority of them have not been proven to be thymus-specific, thymic peptides comprise an effective group of regulators, mediating important immune functions. Thymosin fraction five (TFV) was the first thymic extract shown to stimulate lymphocyte proliferation and differentiation. Subsequent fractionation of TFV led to the isolation and characterization of a series of immunoactive peptides/polypeptides, members of the thymosin family. Extensive research on prothymosin α (proTα) and thymosin α1 (Tα1) showed that they are of clinical significance and potential medical use. They may serve as molecular markers for cancer prognosis and/or as therapeutic agents for treating immunodeficiencies, autoimmune diseases and malignancies. Although the molecular mechanisms underlying their effect are yet not fully elucidated, proTα and Tα1 could be considered as candidates for cancer immunotherapy. In this review, we will focus in principle on the eventual clinical utility of proTα, both as a tumor biomarker and in triggering anticancer immune responses. Considering the experience acquired via the use of Tα1 to treat cancer patients, we will also discuss potential approaches for the future introduction of proTα into the clinical setting.

Thymic peptides for treatment of cancer patients.

Purified thymus extracts (pTE) and synthetic thymic peptides (sTP) are thought to enhance the immune system of cancer patients in order to fight the growth of tumour cells and to resist infections due to immunosuppression induced by the disease and antineoplastic therapy. To evaluate the effectiveness of pTE and sTP for the management of cancer. We searched CENTRAL (The Cochrane Library 2010, Issue 3), MEDLINE, EMBASE, AMED, BIOETHICSLINE, BIOSIS, CATLINE, CISCOM, HEALTHSTAR, HTA, SOMED and LILACS (to February 2010). Randomised trials of pTE or sTP in addition to chemotherapy or radiotherapy, or both, compared to the same regimen with placebo or no additional treatment in adult cancer patients. Two authors independently extracted data from published trials. We derived odds ratios (OR) from overall survival (OS) and disease-free survival (DFS) rates, tumour response (TR) rates, and rates of adverse effects (AE) related to antineoplastic treatments. We used a random-effects model for meta-analysis. We identified 26 trials (2736 patients). Twenty trials investigated pTE (thymostimulin or thymosin fraction 5) and six trials investigated sTP (thymopentin or thymosin α(1)). Twenty-one trials reported results for OS, six for DFS, 14 for TR, nine for AE and 10 for safety of pTE and sTP. Addition of pTE conferred no benefit on OS (RR 1.00, 95% CI 0.79 to 1.25); DFS (RR 0.97, 95% CI 0.82 to 1.16); or TR (RR 1.07, 95% CI 0.92 to 1.25). Heterogeneity was moderate to high for all these outcomes. For thymosin α(1) the pooled RR for OS was 1.21 (95% CI 0.94 to 1.56, P = 0.14), with low heterogeneity; and 3.37 (95% CI 0.66 to 17.30, P = 0.15) for DFS, with moderate heterogeneity. The pTE reduced the risk of severe infectious complications (RR 0.54, 95% CI 0.38 to 0.78, P = 0.0008; I² = 0%). The RR for severe neutropenia in patients treated with thymostimulin was 0.55 (95% CI 0.25 to 1.23, P = 0.15). Tolerability of pTE and sTP was good. Most of the trials had at least a moderate risk of bias. Overall, we found neither evidence that the addition of pTE to antineoplastic treatment reduced the risk of death or disease progression nor that it improved the rate of tumour responses to antineoplastic treatment. For thymosin α(1), there was a trend for a reduced risk of dying and of improved DFS. There was preliminary evidence that pTE lowered the risk of severe infectious complications in patients undergoing chemotherapy or radiotherapy.

Augmentation of mitogen-induced interferon production in lymphocytes by thymosin.

Thymosin (thymosin fraction 5), in combination with mitogen (concanavalin A) augments the amount of interferon produced in human peripheral blood lymphocytes (in vitro studies). In addition, after pretreatment of mice with thymosin (intraperitoneal administration), thymosin augments the mitogen-induced interferon production in mouse lymphocytes in vitro. The produced interferon is immune interferon (IFN-gamma) as determined by sensitivity to pH 2 and lack of neutralization by antibodies to IFN-alpha or IFN-beta. Results indicate that thymic hormones may also participate in the regulation of interferon production.

The β‐thymosins: Intracellular and extracellular activities of a versatile actin binding protein family

The beta-thymosins are N-terminally acetylated peptides of about 5 kDa molecular mass and composed of about 40-44 amino acid residues. The first member of the family, thymosin beta4, was initially isolated from thymosin fraction 5, prepared in five steps from calf thymus. Thymosin beta4 was supposed to be specifically produced and released by the thymic gland and to possess hormonal activities modulating the immune response. Various paracrine effects have indeed been reported for these peptides such as cardiac protection, angiogenesis, stimulation of wound healing, and hair growth. Besides these paracrine effects, it was noted that beta-thymosins occur in high concentration in the cytoplasm of many eukaryotic cells and bind to the cytoskeletal component actin. Subsequently it became apparent from in vitro experiments that they preferentially bind to monomeric (G-)actin and stabilize it in its monomeric form. Due to this ability the beta-thymosins are the main intracellular actin sequestering factor, i.e., they posses the ability to remove monomeric actin from the dynamic assembly and disassembly processes of the actin cytoskeleton that constantly occur in activated cells. In this review we will concentrate on the intracellular activity and localization of the beta-thymosins, i.e., their modulating effect on the actin cytoskeleton.

Thymus lymphocytes in uraemic rats and the effect of thymosin fraction 5 in vivo

Thymus lymphocyte subsets in uraemic rats were studied using monoclonal antibodies. Severe and moderate uraemia was induced in rats, and sham-operated and normal rats were used as the controls. As a result, the weight of the thymus decreased in uraemic rats. As for lymphocyte subsets, the frequency of W3/25+OX8+ decreased and those of W3/25-OX8-, W3/25+OX8- and W3/25- OX8+ relatively increased in uraemic rats. All these changes were more significant in severely uraemic than in moderately uraemic rats. When thymosin fraction 5 (TF5) was administered to severely uraemic rats, the weight of the thymus increased and the lymphocytes subsets normalized. These results suggest that uraemia may cause a maturational impairment of thymus lymphocytes by the depression of thymic hormone secretion.

Thymus lymphocytes in uraemic rats and the effect of thymosin fraction 5 in vitro

Cellular immunity of thymus lymphocytes in uraemic rats was studied. Severe and moderate uraemia was induced in rats, and sham-operated and normal rats were used as the controls. As a result, the response of thymus lymphocytes to concanavalin A (Con A) significantly decreased in severely uraemic rats, but did not change in moderately uraemic rats. However, when the thymus lymphocytes were pretreated with thymosin fraction 5, the response to Con A was ameliorated in severely uraemic rats. There was a significant correlation between the effect of thymosin fraction 5 on Con A response and Con A response of thymus lymphocytes. In addition, serum from severe uraemic rats suppressed the response of normal thymus lymphocytes to Con A. These results indicate that severe uraemia may cause an impairment in maturation of thymus lymphocytes, which can be improved by thymosin fraction 5 in vitro.

Thymosin Fraction‐5 Possesses Antiproliferative Properties in HL‐60 Human Promyelocytic Leukemia Cells

Thymosin fraction-5 (TF5) is a protein preparation of the bovine thymus. TF5 stimulates many assays of T cell-mediated immunity. We found that TF5 substantially suppressed proliferation of the rat C6 glioma and MMQ pituitary adenoma cell lines. Our current research using the promyelocytic cell line HL-60 suggests that TF5 also prevents proliferation of human myeloid leukemia cells. Our objective is the purification and chemical characterization of TF5 peptide components responsible for inhibition of HL-60 proliferative capacity. Using the inhibition of HL-60 cell proliferation, we have chemically characterized TF5 using fast protein liquid chromatography (FPLC), reversed-phase high-performance liquid chromatography (RP-HPLC), and high-performance capillary electrophoresis (HPCE). Vital dye-exclusion, oxidative metabolism of chromogenic dyes, and clonogenic growth profiles were used to determine rates of HL-60 proliferation. Our results identified an approximately 6000 Da component of TF5 capable of inducing HL-60 growth arrest. Synchronized HL-60 cells exposed to TF5 and its various constituents were subjected to cytometric analysis by flow cytometry. TF5-treated HL-60 cells had an increased subdiploid faction (i.e., sub-G1) compared to control cells. TF5 also increased Annexin V staining in randomly cycling HL-60 cells. Thus, a TF5 subfraction possesses growth-suppressive activity for human myeloid neoplasms. Our results indicate that this effect is characterized by at least one hallmark of apoptosis. Future clinical management strategies for certain leukemias may involve the use of thymic peptides.

β‐Thymosins

The development of thymosin beta(4) from a thymic hormone to an actin-sequestering peptide and back to a cytokine supporting wound healing will be outlined. Thymosin fraction 5 consists of a mixture of polypeptides and improves immune response. Starting with fraction 5, several main peptides (thymosin alpha(1), polypeptide beta(1), and thymosin beta(4)) were isolated and tested for biological activity. However, none of the isolated peptides were really thymic hormones. They are all biological important peptides with diverse functions. Polypeptide beta(1) is identical to ubiquitin truncated by two C-terminal glycine residues. Several peptides related to thymosin beta(4) were isolated and sequenced from various species. Large amounts of thymosin beta(4) were found in many cells. It was postulated that the beta-thymosins might have a general function. The identification of a biological function of thymosin beta(4) was tedious. In 1990, Dan Safer and his colleagues recognized that thymosin beta(4) sequesters G-actin. The dissociation constant of the complex in the micromolar range allows for fast binding and release of G-actin. beta-Thymosins are the main intracellular G-actin-sequestering peptides in most vertebrate cells. Thymosin beta(4) is unstructured but folds into a stable conformation on binding to G-actin. It is present in the nucleus as well as the cytoplasm and might be responsible for sequestering nuclear actin. Several biological effects are attributed to thymosin beta(4), oxidized thymosin beta(4), or to ac-SDKP possibly generated from thymosin beta(4). However, very little is known about molecular mechanisms mediating the effects attributed to extracellular beta-thymosins.

History of the Discovery of the Thymosins

From a historical perspective, the studies that led to the isolation and characterization of the thymosins began in earnest in the early 1960s in the laboratory of Abraham White at the Albert Einstein College of Medicine in New York. In a 1966 paper in the Proceedings of the National Academy of Sciences, U.S., we first named these thymic-derived factors "Thymosins." By 1972, the thymosin team had moved to the University of Texas Medical Branch in Galveston (UTMB) where an extremely talented group of young scientists and students succeeded over the next 6 years in preparing and testing a highly active partially purified calf thymus preparation, termed thymosin fraction-5 (TF5), which was amenable for scale-up and suitable for clinical use. In 1974, we received the first IND for a thymic hormone preparation from the FDA to begin a phase-I study with TF5 in children with primary immunodeficiency diseases at the University of California Medical Center in San Francisco. The immunorestorative and potentially life-saving properties of TF5 in clinical medicine were first documented in a landmark paper in 1975 by Drs. Arthur Ammann and Diane Wara in the New England Journal of Medicine. TF5 consists of a family of at least 40 mostly small acidic polypeptides, with molecular weights ranging from 1000 to 15,000 Da. This article will identify the key scientists and the milestones involved in the initial studies with TF5 that have led to the chemical characterization of the thymosins and to translational studies from the lab bench to the clinic.

Presence of a peptide component of thymosin fraction-5 manifesting discrete cytostatic properties in HL-60 human promyelocytic leukemia cells

Thymosin fraction-5 (TF5), an array of small molecular weight peptides present in crude extracts of the adult bovine thymus, contains numerous constituents with demonstrable biological activity. Because TF5 generally enhances immune reactivity in a variety of settings, and additionally restricts proliferation of certain neoplasms, we examined the effects of TF5 on proliferative capacity in the human promyelocytic leukemia cell line HL-60. Vital dye-exclusion, oxidative metabolism of chromogenic dyes, and clonogenic growth profiles were monitored to assess rates of cellular proliferation; our results demonstrate that TF5 restricted HL-60 cell growth, an influence that exhibited comparable potency and efficacy among all three indices. This antiproliferative activity was labile, insofar as medium conditioned in HL-60 cells for 24 h became devoid of the initial growth-suppressive activity after 24-h culture when subsequently administered to naïve cultures. Review of cytoarchitectural traits, chromatin staining by TUNEL, and fluorescent cytometric analyses demonstrated that TF5 failed to elicit apoptosis, however, suggesting that this material instead drove treated cells into growth arrest and an unanticipated cytostasis. Qualitatively similar responses were noted in the human monoblastic leukemia cell line U937. Partial purification of TF5 by FPLC yielded a component containing an antiproliferative activity associated with the ∼1000-Da fraction. These results demonstrate that TF5 contains a sub-fraction possessing a growth-suppressive activity capable of restraining normal proliferation of human myeloid neoplasms via the apparent induction of true cytostasis.

Accumulation of radiolabelled low molecular peptides and proteins in experimental inflammation

This study focuses on evaluating accumulation of the low molecular peptides and proteins labelled with 99mTc in rat inflammatory/infection foci. Peptides (human leukocyte dialysate, HLD; thymosin fraction 5, TF5; aprotinin, APT), and proteins (human IgG, HIG) were labelled with 99mTc using redox polymer. The labelling efficiency was evaluated using paper, TLC and/or column chromatography. Biodistribution of labelled substances was evaluated in rats with Staphylococcus aureus infection or with sterile kaolin suspension inflammation 24 h after abscess induction. Accumulation of 99mTc activity was determinated both by external gamma camera imaging and by counting dissected tissues 4 h after administration. The evaluated peptides and proteins show high labelling efficiency ( 99mTc-HLD > 98%, 99mTc-TF5 > 95%, 99mTc-APT > 98%, 99mTc-HIG > 95%). Usage of redox polymer for labelling increases the stability of 99mTc-labelled substances. The labelling efficiency stays nearly the same (95–98%) after 8 h at least. In experimentally induced inflammation the amount of 99mTc-peptides and 99mTc-HIG activity accumulated is 2.5–6.5 and 5.3–10.6 times higher than in a control tissue. When comparing two types of model inflammations (kaolin- and Staphylococcus-induced ones), the values measured with 99mTc-peptides are more than double than those of kaolin suspension inflammation. The studied low molecular peptides labelled with 99mTc allow rapid localisation of infection foci. 99mTc labelled HIG proved useful for detection of infections and inflammatory lesions.

Avian thymic hormone treatment of peripheral blood mononuclear cells from young chicks stimulates acute graft-versus-host reaction in chicken embryos

Avian thymic hormone (ATH) is a parvalbumin produced by epithelial cells in the thymic cortex of chickens and circulates in the blood on a 5-day cycle. It stimulates precocious development of cell-mediated immunity. The effect of partially purified extracts of thymus (TE) and purified ATH were tested for their effect on the acute graft-versus-host reaction (GVHR). Treatment of chicks for their first 3-days of life did not enhance the acute GVHR produced by their PBMC in 14-day-old embryos. PBMC from 3-day-old chicks were treated in vitro with TE, ATH, thymosin fraction 5 or thymosin α1 for 2 h and injected into 14-day-old embryos. Bone marrow cells and thymic lymphocytes were treated with TE. Only PBMC treated with TE or ATH produced an enhanced acute GVHR. Because ATH targets γδ T cells, the data implicate participation of donor γδ T cells in the acute GVHR.

Thymalfasin for the treatment of chronic hepatitis B

Chronic hepatitis B virus infection is a serious problem because of its worldwide distribution and possible adverse chronic sequelae, such as cirrhosis and hepatocellular carcinoma. Chronic hepatitis B infection is a dynamic state of interactions between the virus, hepatocyte and host immune response. Interferon-Α and direct antiviral agents, such as lamivudine (Epivir®, GlaxoSmithKline), are effective in the therapy of chronic HBV infection but the efficacy is far from satisfactory. Thymalfasin (thymosin Α1; TΑ1, Zadaxin™, SciClone Pharmaceuticals, Inc.) is a 28-amino acid polypeptide produced synthetically but originally isolated from thymosin fraction 5, a bovine thymus extract containing a number of immunologically active peptides. In vitro studies have shown that TΑ1 can influence T-cell production and maturation, stimulate production of Th1 cytokines such as interferon-Γ and interleukin-2, and activate natural killer cell-mediated cytotoxicity. Seven randomized controlled studies on TΑ1 monotherapy in patients with chronic hepatitis B showed that 6 months treatment with TΑ1 (1.6 mg twice-weekly) resulted in a significantly higher sustained response rate than untreated controls. The benefits of TΑ1 therapy is usually not immediately apparent during therapy. There is a trend for complete virological response to increase or accumulate gradually after the end of thymosin therapy. The results of TΑ1 and interferon combination therapy in two open-label trials were also promising. In terms of the mechanisms of action, a combination of TΑ1 and nucleoside or nucleotide analogs is a logical approach in the control of chronic HBV infection and a randomized control study is ongoing.

Thymosins: chemistry and biological properties in health and disease

This paper will review the historical background that has generated our present interest in the actions of the thymosins in biological therapy. It will also discuss the multiple actions of the thymosins in the immune, endocrine and central nervous systems. The isolation from the thymus gland of the thymosins, a family of biologically active molecules with hormone-like properties, was first described in 1966 by AL Goldstein and A White. Since that time, significant progress has been made in understanding the role of the thymosins in immunity and the nature of the growth factors, cytokines and chemokines they modulate. The thymosins include a family of biochemically and functionally distinct polypeptides with clinically important physiological properties. In the early 1970s, preclinical studies establishing the immunorestorative effects of a partially purified thymosin preparation termed thymosin fraction 5 (TF5) provided the scientific foundation for the first clinical trials with TF5 in 1974. TF5 was effective in turning on the immune systems of a number of children with DiGeorge syndrome and other thymic dysplasias. These trials led to further interest in the active components in TF5 and to the chemical characterisation of the biologically active thymosins. Several of these molecules are showing significant promise in the clinic in the areas of cancer, infectious diseases and wound healing.

The Thymosins: Prothymosin α, Parathymosin, and β-Thymosins: Structure and Function

The studies on thymosins were initiated in 1965, when the group of A. White searched for thymic factors responsible for the physiological functions of thymus. To restore thymic functions in thymic-deprived or immunodeprived animals, as well as in humans with primary immuno-deficiency diseases and in immunosuppressed patients, a standardized extract from bovine thymus gland called thymosin fraction 5 was prepared. Thymosin fraction 5 indeed improved immune response. It turned out that thymosin fraction 5 consists of a mixture of small polypeptides. Later on, several of these peptides (polypeptide beta 1, thymosin alpha 1, prothymosin alpha, parathymosin, and thymosin beta 4) were isolated and tested for their biological activity. The research of many groups has indicated that none of the isolated peptides is really a thymic hormone; nevertheless, they are biologically important peptides with diverse intracellular and extracellular functions. Studies on these functions are still in progress. The current status of knowledge of structure and functions of the thymosins is discussed in this review.