key: cord-0729207-c699hhs8
authors: Li, Juan; Liu, Chun Hui; Wang, Feng Shan
title: Thymosin alpha 1: Biological activities, applications and genetic engineering production
date: 2010-08-08
journal: Peptides
DOI: 10.1016/j.peptides.2010.07.026
sha: af8a40ac298182ece4b5f9a2973526a4e72d817f
doc_id: 729207
cord_uid: c699hhs8

Thymosin alpha 1 (Tα1), a 28-amino acid peptide, was first described and characterized from calf thymuses in 1977. This peptide can enhance T-cell, dendritic cell (DC) and antibody responses, modulate cytokines and chemokines production and block steroid-induced apoptosis of thymocytes. Due to its pleiotropic biological activities, Tα1 has gained increasing interest in recent years and has been used for the treatment of various diseases in clinic. Accordingly, there is an increasing need for the production of this peptide. So far, Tα1 used in clinic is synthesized using solid phase peptide synthesis. Here, we summarize the genetic engineering methods to produce Tα1 using prokaryotic or eukaryotic expression systems. The effectiveness of these biological products in increasing the secretion of cytokines and in promoting lymphocyte proliferation were investigated in vitro studies. This opens the possibility for biotechnological production of Tα1 for the research and clinical applications.

Thymosin alpha 1 (T␣1), a biologically active peptide consisting of 28 amino acid residues, was first described and characterized by Goldstein et al. [34] . The research process of T␣1 began with the study on the thymus, which is an important vital organ for homeostatic maintenance of peripheral immune system [48] . In 1966, Goldstein et al. [35] first isolated and described a lymphocytopoietic factor from calf thymus, which was termed "thymosin". The multiple action of thymosin on the immune, endocrine and central nervous systems was revised by Goldstein and Badamchian [32] . Further purification of this factor led to the isolation of a heat-stable acetone-insoluble preparation, termed thymosin fraction 5 (TF5), which could induce T cell differentiation, enhance immunological function [36] and induce apoptosis of neuroendocrine tumor cells [72] . The promising results seen with TF5 provided the scientific rationale to further isolate and characterize the molecules in TF5 responsible for the reconstitution of T-cell immunity. Hence, T␣1 was first purified from TF5 in 1977 [34] and has been found to be 10-1000 times as active as TF5 evaluated in vivo and in vitro [47] .

T␣1 is the asparaginyl endopeptidase cleavage product of prothymosin ␣ (ProT␣), an acidic nuclear protein consisting of 109 amino acid residues [10] . T␣1 is a highly conserved acid peptide, ubiquitously existing in lymphoid tissues such as spleen and lymph nodes, non-lymphoid tissues such as lungs, kidneys, and brain, but mainly existing in thymus gland [33] , especially in the thymic epithelial cells. Interestingly, the secretion of T␣1 is not modulated by other hormones or releasing factors [54] . As a potent biological response modifier (BRM), T␣1 has intensive clinical applications. In the first randomized double-blind Phase II trial of T␣1 carried out by Schulof et al. [68] , administration of synthetic T␣1 to postradiotherapy patients with non-small cell lung cancer exhibited significant improvements in relapse-free and overall survival, which was most pronounced in patients with nonbulky tumors. Now T␣1 is in clinical trials worldwide for the treatment of several types of cancer, hepatitis B virus (HBV) and hepatitis C virus (HCV) infections, which connect closely with hepatocellular carcinoma (HCC) [77] . Additionally, T␣1 shows remarkable effects in the treatment of other diseases such as severe sepsis [87, 43] , acute respiratory distress syndrome (ARDS) [38] , severe acute respiratory syndrome (SARS) [23] , gastrointestinal and systemic infectious disorders [39] , and spontaneous peritonitis in individuals with cirrhosis [49] .

Because of the extensive applications of T␣1, there is an increasing need to produce T␣1 in larger quantities to keep up with the growing clinical demand. Besides isolated from calf thymus, bioactive T␣1 can be obtained by solid-phase synthesis [78] or genetic engineering [80] , but T␣1 currently used in clinic is entirely solidphase synthesized polypeptide, with chemical features identical to the human T␣1 [40] . Recently, genetic engineering expression of T␣1 in different hosts including Escherichia coli, Pichia pastries and plants [55, 14] has attracted more attention due to its potential for producing low cost and bioactive T␣1.

In this review, we briefly describe the biological activities of T␣1 and discuss the current applications of T␣1 in cancer and infectious diseases. Furthermore, we summarize ways of genetic engineering production of this peptide, which maybe provide a conceptual framework for future studies to improve the quality and the yield of T␣1 for different fields of research and clinical applications.

Many studies have been performed to identify the immunoregulatory activity of T␣1 in vitro and in vivo. Evidence has shown that T␣1 increased the efficiency of T cell maturation [1] , stimulated precursor stem cell differentiation into the CD4 + /CD8 + T cells [57] and balanced CD3/CD4 + /CD8 + T cells of peripheral blood mononuclear cells (PBMCs) [84] . By stimulating natural killer (NK) cells and cytotoxic lymphocytes (CD8 + T cell), T␣1 could directly kill virally infected cells [67] . By activating dendritic cells (DCs), T␣1 was able to protect immunocompromised mice from death caused by aspergillosis [62] . T␣1 stimulated a significant increase of IL-2 and led to a decrease in the Th2 cytokines such as IL-4 and IL-10 in patients with chronic HCV [67] . Besides, T␣1 remarkably decreased the severity of severe acute pancreatitis by having a negative effect on serum levels of IL-1␤ and tumor necrosis factor-alpha (TNF-␣) [84] . T␣1 also upregulated specific cytokine receptors such as high-affinity IL-2 cytokine receptors [42] .

Not only activating immuno-effector cells or modulating cytokines expression, T␣1 also directly exerted its effects on target cells. It could increase the expression of MHC I [30] and tumor antigens [25] , directly depress viral replication [4] , and increase expression of viral antigens on the surface of target-infected cells [24] , making them more visible to the immune system and less prone to escape from immunosurveillance.

Although the observations of the T␣1 potential immunoregulatory effects are clearly evident, what is not clear is the mechanisms of action on the immune system. It was reported that T␣1 could directly modulate the expression of cytokine genes, MHC class I, MHC class II related genes as well as a significant number of new genes, acting as immune system regulators [26] . Naylor and his colleagues demonstrated that genes of major histocompatability proteins, costimulatory molecules, chemokines and cytokines, and their receptors were upregulated in both T cells and monocytes exposed to T␣1 [54] , indicating that there were multiple targets for its immune-enhancing activity. As illustrated in Fig. 1 , T␣1-mediated stimulation of intracellular signaling pathways included mitogen-activated protein kinase (MAPK) transduction pathways [71] and TNF-␣ receptor-associated factor 6 (TRAF6) signal pathway by activating I-kappa B kinase (IKK) [88] . T␣1 has also been found to induce IL-6, IL-10 and IL-12 expression via IRAK4/1/TRAF6/PKC/IKK/NF-B and TRAF6/MAPK/AP-1 pathways [56] . These pathways are shared by many cytokines, which predict potential synergy between T␣1 and cytokines. T␣1 was able to prime DC for antifungal Th1 resistance through Toll-like receptor 9 (TLR9)/myeloid differentiation factor 88 (MyD88)-dependent signaling [62] . Besides, DCs could also be primed by T␣1-induced activation of p38 MAPK, NF-B pathways [83] . Activated plasmacytoid DCs (pDC) led to the activation of interferon regulatory factor 7 and the promotion of the IFN-␣/IFN-␥-dependent effector path- 

T␣1 has been shown to decrease tumor cell growth both in vitro and in vivo and has been demonstrated therapeutic usefulness in several types of cancer (Table 1) . T␣1 was observed to exhibit anti-proliferative effects on HepG2 human hepatoma cells and SPC-A-1 lung adenocarcinoma cells in vitro assays [60] . To explore the anti-metastatic/antitumor activity of T␣1, it was subcutaneously injected into BALB/c-mice, which significantly reduced liver and lung metastases and decreased local tumor growth [6] . Moody et al. investigated the effects of T␣1 on mammary carcinogenesis in fisher rats and found that T␣1 could reduce mammary carcinoma incidence and prolong survival time [52] . In another breast adenoma model, T␣1 increased the survival time in female C3(1)SV40T antigen transgenic mice and fisher rats, but it remained to be determined whether the immune response also increased or not [53] . The antitumor activity of T␣1 was most effective when the lung adenomas were small, which was based on studies proformed by Moody who gave T␣1 daily to A/J mice bearing lung adenoma [53] . T␣1 may fight against tumors through either stimulating the immune system or directly inhibiting the proliferation of tumor cells.

T␣1 in combination with other BRMs or chemotherapy agents also displays good effects in reducing tumor burden and progression. The plasmid-liposome complex containing the cDNA of human T␣1 and IFN 1 was injected into ICR mice, and the dual-gene plasmid-liposome complex showed stronger inhibitory effect on the growth of tumor than the single gene of T␣1 or IFN 1, which might attribute to indirect and additive induction of apoptosis of tumor cells by the increased expression of T␣1 and IFN 1 [12] . In DHD-K12 colorectal cancer model, combination of 5-FU, IL-2 and T␣1 could dramatically increase survival rates as well as control tumor metastasis [26] . Similarly, compared with Carmustine (BCNU) monotherapy, intraperitoneal injection of T␣1 and BCNU to adult Long Evans rats bearing glioblastoma could significantly lower the tumor burdens and increase the cure rates [73] . Since the cascades and feedback networks of immune responses, the combination of immunoactive molecules that affected different immune effector cells resulted in a stimulation of the immune response significantly stronger than that evoked by single treatments. This could contribute in helping explain the mechanisms of the significance of combination therapy.

Several reports showed that T␣1 had protective effects against oxidative damage. T␣1 had a positive influence on liver superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activity and thereby limited free radical damages to hepatic tissue [5] . Similarly, it was reported that T␣1 could ameliorate streptozotocin-induced pancreatic lesions and diabetes by reducing malondialdehyde (MDA), increasing GSH level and enhancing the activities of both SOD and catalase (CAT), suggesting that T␣1 treatment could greatly enhance the overall antioxidative capability of pancreatic tissues [61] .

T␣1 possesses the ability of influencing the central nervous system [68, 70] . Its modulatory effect on the excitatory synaptic transmission in cultured hippocampal neurons was documented [81] . Similarly, when it was combined with chemotherapeutics in treating cancers, T␣1 could prevent patients from chemotherapyinduced neurotoxicities [2] . Moreover, T␣1 has potent effects in promoting endothelial cell migration, angiogenesis as well as wound healing [50] .

Patients with cancer are often accompanied with significant deficiencies in cellular immunity. In addition, standard treatments for cancer usually induce significant depression of the immune response. T␣1 has been demonstrated to decrease tumor cell growth both in vitro and in vivo and has therapeutic effect in several types of cancer. In advanced lung or advanced breast cancer, T␣1 combined with chemotherapy could prevent patients from chemotherapy-induced neurotoxicities [2] . In a Phase II multicenter, randomized open-label study, different dose levels of T␣1 in combination with Dacarbazine (DTIC) chemotherapy were given to patients with stage IV melanoma. Reported results show that the combination therapy tripled the overall response rate and extended overall survival by nearly 3 months compared with patients treated with DTIC, combined with IFN-␣ [8] . More recently, in patients with unresectable HCC, transarterial chemoembolization (TACE) combined with T␣1 resulted in numerically higher rates of survival and tumor response, including transplant candidacy, with fewer bacterial infections, than TACE alone [29] .

Obviously, significant tumor growth inhibition and survival rate increase were achieved in different human tumor models when T␣1 was combined with other treatment modalities. It can be concluded that combinatorial therapies, in which T␣1 represents one important mediator, are effective therapeutic strategy against tumors and will be the key focus for the use of T␣1 in treating cancers in the future.

Chronic HBV infection is a serious clinical problem because of its worldwide distribution and potential adverse sequelae, such as cirrhosis and hepatocellular carcinoma [44] . T␣1 has been approved for the treatment of hepatitis B in many countries worldwide with a significantly increasing virological response over time after therapy [58] . Most of the studies have evaluated the efficacy of T␣1 in the treatment of HBeAg-positive and HBeAg-negative chronic hepatitis B. For instance, administering T␣1 either 0.8 mg or 1.6 mg to 316 Japanese patients with HBeAg-positive chronic hepatitis B showed HBeAg seroconversion in 18.8% and 21.5% at 48 weeks after the end of treatment, respectively [37] . Similarly, administering T␣1 1.6 mg to Chinese patients with HBeAg-negative chronic hepatitis B twice weekly showed a complete response, defined as normalization of alanine transaminase (ALT) and undetectable HBV DNA by PCR assay, in 11 of 26 patients (42.3%) at 6 months after the end of treatment [86] . Zhang et al. searched materials from different databases and analyzed eight trials using meta analysis. They found that lamivudine and T␣1 combination treatment was particularly prominent than lamivudine monotherapy in terms of ALT normalization rate, virological response rate and HBeAg seroconversion rate [89] . Conversely, Lee et al. [41] revealed that combining T␣1 and lamivudine did not display a better benefit to virological and biochemical response than the lamivudine monotherapy. Maybe the small trial scale led to the divergent results.

As a monotherapy, T␣1 does not seem useful in treating HCV infection, which is confirmed by a randomized, double-blind, placebo-controlled trial [3] . However, combination therapy of T␣1 and pegylated interferon ␣2a (peg-IFN-␣2a) could effectively suppress viral replication in difficult-to-treat hepatitis C patients. In addition, T␣1 was well tolerated with no significant adverse effects observed [66] . Approximately 50% of treatment-naive HCV patients failed to achieve a sustained virologic response (SVP) with standard peg-IFN and ribavirin therapy [21] , so a triple combination therapy with peg-IFN-␣2a, ribavirin and T␣1 has been developed and proved to be a safe [7] and effective [59] treatment option for difficult-to-treat HCV patients who are refractory to prior conventional treatment.

Human immunodeficiency virus (HIV) specially targets cells that express CD4, such as macrophages, DCs and CD4 + T cells. When the virus becomes lymphotropic, it begins to infect CD4 + T cells efficiently followed by significantly declined antibody class switching. Furthermore, CD8 + T cells are not stimulated as effectively, facilitating the escape of the virus from immune control and the collapse of the whole immune system [51] . Since significant immune responses play an important role in the prevention of infection with human HIV, it is thought that the induction of strong immune responses especially CTL responses against HIV-1 could be important to prevent the onset of acquired immune deficiency syndrome (AIDS) [74] . One study has suggested that combination of T␣1, zidovudine (AZT) and IFN-␣ resulted in a significant increase in the number and function of CD4 + T cells and a reduction in HIV titers [27] . Another interesting finding was provided by Chadwick et al. [9] , who studied the safety and efficacy of T␣1 in combination with highly active antiretroviral therapy (HAART) in stimulating immune reconstitution. The results demonstrated that T␣1 appeared to be very well tolerated and could dramatically increase the levels of signal joint T cell receptor excision circles (sjTREC) in patients with advanced HIV disease. However, longer treatment duration of T␣1 in augmenting the immune reconstitution needs further investigation.

From the natural source, T␣1 can only be obtained in tiny quantities. To obtain larger amounts of this peptide, literally tons of fresh frozen calf thymus tissue and acetone are required [31] . Furthermore, heterogeneous allergens introduced by manufacturing process limit its availability for research and medical applications. Solid phase synthesis has permitted scientists to synthesize and purify T␣1 to allow human clinical trials [78, 79] with the advantages of simplicity, ease of operation, general efficiency and lack of endotoxins and DNA contaminations. However, difficult sequences T␣1 bears and the high number of protecting groups required to assemble the peptide may give final low yields, insufficient purity and high expenses. A recent report revealed that combination of the side-chain anchoring approach with the hydrophilicity of the totally PEG-based resin facilitated the synthesis of T␣1 in high purity and high yields [28] .

With the advancement in genetic engineering, bioactive T␣1 can be expressed in prokaryotic and eukaryotic expression systems, which are cost-effective alternative approaches to produce biotechnical drugs. These products including T␣1, fusion proteins and concatemers produced in E. coli, P. pastoris and plants were all soluble expressed, which could escape from refolding from inclu- Fig. 3 . Construction of the concatemer gene. A T-vector containing the T␣1 gene (in red) was digested with BamHI/XhoI and BglII/XhoI respectively. When digested with BglII and BamHI, the two fragments had identical termini and could be ligated with T4 DNA ligase subsequently forming a new sequence GGATCT, which could not be digested by neither BamHI nor BglII. Therefore, a plasmid containing double T␣1 genes was constructed and could not be destroyed when the concatemer T˛1 gene of 4 repeats was constructed. Thus, the plasmid containing concatemer T˛1 gene of 4 repeats and 6 repetats could also be constructed [90] . (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.) sion bodies (see Fig. 2 ). But isolation and purification of them with a high purity is difficult and applying them to clinical treatment has not come true. A number of studies of using genetic engineering to gain bioactive T␣1 are summarized as follows.

E. coli is one of the earliest and most widely used hosts for the production of heterologous proteins [75] . It has the advantages of rapid growth and expression, easy culture and high yields. However, E. coli is not the system of choice for expressing disulfide rich proteins and proteins that require post-translational modifications [17] . With the characteristics of small molecular weight and needless post-translational modifications, T␣1 is suitable to be expressed by E. coli system. Several strategies for the expression of T␣1, T␣1-BRMs fusion proteins, T␣1 concatemer and so on using E. coli expression system have been reported.

Generally, small peptides are difficult to be overexpressed directly in E. coli since they can be quickly degraded by cellular proteases. The use of protease-deficient host strains and fusion tags, such as his-tag and thioredoxin can help to avoid non-specific proteolytic degradation and facilitate purification. Following this approach, the synthesized human T␣1 gene was inserted into pET-28a (+) plasmid and then inductively expressed as a soluble form in E. coli BL21, which is a protease-deficient host strain. Compared with other expression systems, the BL21/pET-28a system provided the highest expression level of fusion protein, which amounted to 70% of total expressed proteins [13] . Furthermore, T␣1 gene was inserted into pET32b (+) and expressed with thioredoxin in E. coli strain ER2566. After proteolytic cleavage and chemical acetylation, the resultant T␣1 was purified by reversed-phase highperformance liquid chromatography (RP-HPLC) with the yield of 29 mg per litre of bacterial culture. This method is simple, costeffective and suitable for large-scale production of T␣1 [18] .

Since improved control of tumor growth can be observed when tumor-bearing mice were treated with T␣1 and high doses of IL-2 [46] , combination therapies have performed and have been proved to be effective in inhibiting tumor growth and in controlling infectious diseases especially in the immunocompromised host. Thus, the expression of fused molecules of T␣1 and other BRMs which have synergistic effect with T␣1 was investigated. T␣1 and cBLyS, a soluble B-cell lymphocyte stimulator amplifying the humoral response, were fused with a flexible linker sequence and expressed in E. coli. This bifunctional lymphokine was useful in the treatment of various immunodeficiency syndromes and served as an immunomodulator to enhance the host's response to vaccination [69] . The fusion protein of T␣1 and consensus IFN␣ (IFN␣-con), which was soluble and amounted to more than 20% of total proteins of E. coli, showed higher antiviral effect than IFN␣ and the activity in promoting lymphocyte proliferation was similar to commercial T␣1 [45] .

It is difficult to extract and purify T␣1 from the fermentation broth since its molecular weight is small. The concatemer strategy maybe partially solve the problem of low expression and the difficulty of purification by increasing the size of the target molecule. A concatemer T˛1 gene of 6 repeats was constructed according to the E. coli codon usage preference, ligated with expression vector pET-22b (+) and transformed into E. coli BL21 (DE3) [90] . The T␣1 monomer was successfully released by hydroxylamine inci-sion after concatemer purification, and its activity in promoting mice splenic lymphocyte proliferation was approximatively identical to the natural T␣1. The intimate process of construction of the concatemer T˛1 gene is described in Fig. 3 .

In addition, a concatemer T˛1 gene of 4 repeats was synthesized and successfully expressed in E. coli in a soluble form. Preliminary results demonstrated that the concatemer protein also had the activity in stimulating mouse spleen lymphocyte proliferation [15] .

It is a common knowledge that E. coli lacks efficient posttranslational modification systems for modifying exogenous proteins. However, Fang et al. [20] found that the fusion protein of T␣1 and ribosomal protein L12 was partly N ␣ -acetylated when expressed in E. coli and this modification was performed by RimJ, which is the N terminal acetyltransferase that modifies the ribosomal protein S5 [85] and acts as an ribosome assembly factor [65] . This enlightens us that fully acetylated T␣1 can be obtained by coexpressing with RimJ. However, little is known about the pathway by which this fusion protein is N ␣ -acetylated. The previous reports that the activity of none or partly N ␣ -acetylated T␣1 is similar to the natural one [80] illustrated that N ␣ -acetylation of T␣1 could influence the stability of the peptide instead of the bioactivity in vivo.

Based on the above research findings, it can be concluded that T␣1 is suitable to be expressed by E. coli expression system. To improve the expression efficacy, the following measures may be meaningful: (i) choosing E. coli usage preference codons; (ii) using different promoters to regulate expression; (iii) using proteasedeficient host strains.

Yeasts are attractive hosts for the production of heterologous proteins for providing post-translational modifications and generating stable cell lines via homologous recombination [16] . Some examples of expressing T␣1 in yeast expression system are presented as follows.

Chen et al. [11] successfully constructed an effective yeast expression system for T␣1 in which pYES2-T␣1 plasmid was transformed into INVSc1 yeast host strain, and T␣1 expressed by this system could improve the level of CD8 + cells in BALB/c mice treated with cyclophosphamide in advance.

Fusion expression of T␣1 and other BMRs in P. pastoris are also reported. IFN␣2b exhibits synergic effects with T␣1 in the treatment of hepatitis B and hepatitis C. The fusion protein of IFN␣2b and T␣1 linked by different lengths of (Gly-Gly-Gly-Gly-Ser)n (n = 1-3) were expressed in P. pastoris and exhibited both antiviral activity of IFN␣2b and immunomodulatory activity of T␣1 estimated in vitro [82] . Thymopentin (TP5) not only acts as an immunomodulatory factor in cancer chemotherapy, but is also a potential chemotherapeutic agent in the human leukemia therapy [19] . However, extremely short half-life in vivo (30 s) [76] greatly restricts its clinical applications. In this sense, a T␣1-TP5 fusion gene was synthesized, inserted into vector pGAPZ␣A and expressed in P. pastries by our research team [22] . The T␣1-TP5 fusion peptide displayed higher activity than T␣1 and TP5 in promoting the phagocytosis of macrophages and the proliferation of Kunming mouse splenocytes.

Plants are also used for the production of T␣1 in the form of monomer or concatemer [55, 14] . Recently, the concatemer T˛1 gene of 4 repeats was introduced and successfully expressed in transgenic tomatoes. The bioactivity of concatemer protein for stimulating proliferation of mice splenic lymphocytes in vitro was stronger than that synthesized artificially or T␣1 concatemer pro-tein expressed in the E. coli system, but the underlying reasons were unclear and required further investigation [14] .

These examples demonstrate that bioactive T␣1 can be obtained by genetic engineering. With great efforts are being made, such as improving the quality, functionality, purity and yield of T␣1 products, it can be expected that over the next few years they will find their way into the clinic.

In addition to immunomodulatory activity, T␣1 owns the ability of influencing the central nervous system and regulating endocrine system. It is very likely that due to its pleiotropic biological activities, T␣1, either alone or combined with other treatment strategies, will have a broader spectrum of applications for successful treatment of various diseases in clinic. Up to now, chemical synthesis is the only effective way to produce T␣1 for clinical therapy. Genetic engineering is an attractive alternative route of expressing bioactive T␣1, but at present, it offers no higher purity of T␣1 compared with chemical synthesis. Recently, gene expression of T␣1 concatemer and fusion proteins have become a major research focus, which are effective strategies for facilitating purification, increasing production and reducing production costs. It is believed that the rapid development of biotechnology may allow application of T␣1 products obtained by genetic engineering in clinic in the future.

Tlymphocyte maturation: cell surface markers and immune functions induced by T-lymphocyte cell-free products and thymosin polypeptides

Primary assessment of treatment effect of thymosin alpha1 on chemotherapy-induced neurotoxicity

A double-blind, placebocontrolled pilot trial of thymosin alpha 1 for the treatment of chronic hepatitis C

Thymosinalpha 1 plus interferon-alpha for naive patients with chronic hepatitis C: results of a randomized controlled pilot trial

Thymosin alpha 1 attenuates lipid peroxidation and improves fructose-induced steatohepatitis in rats

Thymosin alpha(1) applications augments immune response and down-regulates tumor weight and organ colonization in BALB/c-mice

Studies of therapy with thymosin alpha 1 in combination with pegylated interferon alpha2a and ribavirin in nonresponder patients with chronic hepatitis C

A large firstline randomized dose-finding, phase II study on thymosin ␣ (IFN␣) compared to DTIC plus IFN␣ in stage IV melanoma tumor response and survival results (ASCO meeting Abstracts)

A pilot study of the safety and efficacy of thymosin alpha 1 in augmenting immune reconstruction in HIV-infected patients with low CD4 counts taking highly active antiretroviral therapy

Roles of thymosins in cancers and other organ systems

Construction and applications of a yeast expression system for thymosin alpha 1

Liposomal plasmid DNA encoding human thymosin alpha and interferon omega potently inhibits liver tumor growth in ICR mice

Overexpression of soluble human thymosin alpha 1 in Escherichia coli

Expression of thymosin alpha1 concatemer in transgenic tomato (Solanum lycopersicum) fruits

Expression and analysis of thymosin alpha1 concatemer in Escherichia coli

Expression of heterologous proteins in pichia pastoris: a useful experimental tool in protein engineering and production

Production of recombinant proteins by microbes and higher organisms

Production of thymosin alpha1 via non-enzymatic acetylation of the recombinant precursor

Thymopentin (TP5), an immunomodulatory peptide, suppresses proliferation and induces differentiation in HL-60 cells

RimJ is responsible for N ␣ -acetylation of thymosin ␣1 in Escherichia coli

Peginterferon alfa-2a plus ribavirin for chronic hepatitis C vious infection

Expression of thymosin alpha1-thymopentin fusion peptide in pichia pastoris and its characterization

Clinical investigation of outbreak of nosocomial severe acute respiratory syndrome

Thymosin alpha1: a historical overview

Thymosin alpha 1: from bench to bedside

Thymosin alpha(1) in combination with cytokines and chemotherapy for the treatment of cancer

Combination treatment with zidovudine, thymosin alpha 1 and interferon-alpha in human immunodeficiency virus infection

Optimized Fmoc solidphase synthesis of Thymosin alpha1 by side-chain anchoring onto a PEG resin

A randomized controlled trial of thymalfasin plus transarterial chemoembolization for unresectable hepatocellular carcinoma

Thymosin-alpha1 regulates MHC class I expression in FRTL-5 cells at transcriptional level

History of the discovery of the thymosins

Thymosins: chemistry and biological properties in health and diseases

From lab to bedside: emerging clinical applicationss of thymosin alpha 1

Thymosin alpha1: isolation and sequence analysis of an immunologically active thymic polypeptide

Preparation, assay, and partial purification of a thymic lymphocytopoietic factor (thymosin)

Purification and properties of bovine thymosin

The efficacy and safety of thymosin alpha-1 in Japanese patients with chronic hepatitis B; results from a randomized clinical trial

Immunoregulation of thymosin alpha 1 treatment of cytomegalovirus infection accompanied with acute respiratory distress syndrome after renal transplantation

The rapidly expanding therapeutic role of thymosin alpha-1 in the management of gastrointestinal and systemic infectious disorders

Thymosin alphal: a promising molecule for important clinical applications

Combination therapy of thymosin alpha-1 and lamivudine for HBeAg positive chronic hepatitis B: a prospective randomized, comparative pilot study

Thymosin alpha 1 modulates the expression of high affinity interleukin-2 receptors on normal human lymphocytes

A new immunomodulatory therapy for severe sepsis: ulinastatin plus thymosin {alpha} 1

Thymalfasin (thymosin-alpha 1) therapy in patients with chronic hepatitis B

Expression and activity analysis of interferonalpha-con and thymosin-alpha1

Biochemotherapy with thymosin alpha 1, interleukin-2 and dacarbazine in patients with meta-static melanoma: clinical and immunological effects

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

Thymic involution and immune reconstitution

A clinical study of thymosin alpha1 as an auxiliary in treating spontaneous peritonitis in patients with liver cirrhosis

Thymosin alpha 1 stimulates endothelial cell migration, angiogenesis, and wound healing

Cellular immune responses to HIV

Thymosin alpha1 inhibits mammary carcinogenesis in Fisher rats

Thymosin alpha 1 as a chemopreventive agent in lung and breast cancer

Immunopharmacology of thymosin alpha 1 and cytokine synergy

High expression of thymosin alpha 1 by injecting recombinant PVX vector into the tomato fruit

Signaling pathways leading to the activation of IKK and MAPK by thymosin alpha 1

Effects of thymic polypeptides on the thymopoiesis of mouse embryonic stem cells

Reviews for APASL guidelines: immunomodulator therapy of chronic hepatitis B

Efficacy of triple therapy with thymalfasin, peginterferon alpha-2a, and ribavirin for the treatment of hispanic chronic HCV nonresponders

Proliferative and anti-proliferative effects of thymosin alpha 1 on cells are associated with manipulation of cellular ROS levels

Intraperitoneal co-administration of thymosin alpha-1 ameliorates streptozotocin-induced pancreatic lesions and diabetes in C57BL/6 mice

Thymosin alpha 1 activates dendritic cells for fungal Th1 resistance through Toll-like receptor signaling

Thymosin alpha1: an endogenous regulator of inflammation, immunity, and tolerance

Thymosin alpha 1 activates dendritic cell tryptophan catabolism and establishes a regulatory environment for balance of inflammation and tolerance

Suppression of a cold-sensitive mutation in ribosomal protein S5 reveals a role for RimJ in ribosome biogenesis

Combination therapy of thymalfasin (thymosin-alpha 1) and peginterferon alfa-2a in patients with chronic hepatitis C virus infection who are non-responders to standard treatment

Thymalfasin for the treatment of chronic hepatisis C infection

A randomized trial to evaluate the immunorestorative properties of synthetic thymosin-alpha 1 in patients with lung cancer

Construction and expression of a new fusion protein, thymosin alpha 1-cBLyS

The peptide molecular links between the central nervous and the immune systems

Involvement of mitogen-activated protein kinases in the signal transduction pathway of bone marrow-derived macrophage activation in response to in vitro treatment with thymosin alpha 1

Thymosin fraction 5 inhibits the proliferation of the rat neuroendocrine MMQ pituitary adenoma and C6 glioma cell lines in vitro

Potential role of thymosin-alpha1 adjuvant therapy for glioblastoma

Induction of immune responses to a human immunodeficiencyvirus type 1 epitope by novel chimeric influenza viruses

Overview of bacterial expression systems for heterologous protein production: from molecular and biochemical fundamentals to commercial systems

Short in vitro halflife of thymopoietin 32-36 pentapeptide in human plasma

Incidence of hepatocellular carcinoma among individuals with hepatitis B virus infection identified using an automated data algorithm

Automated synthesis of thymosin alpha 1

Solid-phase synthesis of thymosin alpha 1 using tertbutyloxycarbonylaminoacyl-4-(oxymethyl)phenylacetamidomethyl-resin

Production of biologically active N␣-desacetyl thymosin ␣1 in Escherichia coli through expression of a chemically synthesized gene

Thymosin alpha-1 modulate excitatory synaptic transmission in cultured hippocampal neurons in rats

Construction, expression and characterization of human interferon alpha 2b-(G4S)n-thymosin alpha 1 fusion proteins in Pichia pastoris

Thymosin alpha 1 modulates dendritic cell differentiation and functional maturation from human peripheral blood CD14+ monocytes

Thymosin alpha 1 improves severe acute pancreatitis in rats via regulation of peripheral T cell number and cytokine serum level

Cloning and nucleotide sequencing of the genes rimI and rimJ which encode enzymes acetylating ribosomal proteins S18 and S5 of Escherichia coli K12

A randomized, controlled, clinical study of thymosin alpha-1 versus interferon-alpha in patients with chronic hepatitis B lacking HBeAg in China

Evaluation of the efficacy of thymosin alpha 1 in the treatment of sepsis: a systemic review

Activation of IKK by thymosin alpha 1 requires the TRAF6 signalling pathway

Treatment with lamivudine versus lamivudine and thymosin alpha-1 for e antigen-positive chronic hepatitis B patients: a meta-analysis

Expression and hydroxylamine leavage of thymosin alpha 1 concatemer