The Protective Effects of Water-Soluble Alginic Acid on the N-Terminal of Thymopentin.

Thymopentin (TP5) has exhibited strong antitumor and immunomodulatory effects in vivo. However, the polypeptide is rapidly degraded by protease and aminopeptidase within a minute at the N-terminal of TP5, resulting in severe limitations for further practical applications. In this study, the protective effects of water-soluble alginic acid (WSAA) on the N-terminal of TP5 were investigated by establishing an H22 tumor-bearing mice model and determining thymus, spleen, and liver indices, immune cells activities, TNF-α, IFN-γ, IL-2, and IL-4 levels, and cell cycle distributions. The results demonstrated that WSAA+TP5 groups exhibited the obvious advantages of the individual treatments and showed superior antitumor effects on H22 tumor-bearing mice by effectively protecting the immune organs, activating CD4+ T cells and CD19+ B cells, and promoting immune-related cytokines secretions, finally resulting in the high apoptotic rates of H22 cells through arresting them in S phase. These data suggest that WSAA could effectively protect the N-terminal of TP5, thereby improving its antitumor and immunoregulatory activities, which indicates that WSAA has the potential to be applied in patients bearing cancer or immune deficiency diseases as a novel immunologic adjuvant.

Improving the nutritional value and bioactivity of soybean meal in solid-state fermentation using Bacillus strains newly isolated from the gut of the termite Termes propinquus.

The present study aimed to isolate and characterize proteolytic Bacillus spp. from termite guts to test the possibility of application for improving the nutritional value and bioactivity of fermented soybean meal (FSBM). Aerobic endospore-forming bacteria were isolated from the gut of the termite Termes propinquus. Ten isolates with high levels of soy milk degradation were selected and tested for extracellular enzyme production. Among them, two isolates, Tp-5 and Tp-7, exhibited all tested hydrolytic enzyme activities (cellulase, xylanase, pectinase, amylase, protease, lipase and phytase), weak alpha hemolytic and also antagonistic activities against fish pathogenic species of Aeromonas and Streptococcus. Both phylogenetic and biochemical analyses indicated that they were closely related to Bacillus amyloliquefaciens. During solid-state fermentation of SBM, Tp-5 and Tp-7 exhibited the highest protease activity (1127.2 and 1552.4 U g-1, respectively) at 36 h, and the resulting FSBMs showed a significant increase in crude protein content and free radical-scavenging ability (P < 0.05), as well as an improvement in the composition of amino acids, metabolites and other nutrients, while indigestible materials such as fiber, lignin and hemicellulose were decreased. The potential strains, especially Tp-7, improved the nutritional value of FSBM by their strong hydrolytic and antioxidant activities, together with reducing antinutritional components.

Targeting the TLR2 Receptor With a Novel Thymopentin-Derived Peptide Modulates Immune Responses.

The innate and adaptive immune systems act in concert to protect us from infectious agents and other harmful substances. As a state of temporary or permanent immune dysfunction, immunosuppression can make an organism more susceptible to infection, organ injury, and cancer due to damage to the immune system. It takes a long time to develop new immunomodulatory agents to prevent and treat immunosuppressive diseases, with slow progress. Toll-like receptor 2 (TLR2) agonists have been reported as potential immunomodulatory candidates due to their effective activation of immune responses. It has been demonstrated that thymopentin (TP5) could modulate immunity by binding to the TLR2 receptor. However, the fairly short half-life of TP5 greatly reduces its pharmacological potential for immunosuppression therapy. Although peptide cathelicidin 2 (CATH2) has a long half-life, it shows poor immunomodulatory activity and severe cytotoxicity, which seriously hampers its clinical development. Peptide hybridization is an effective approach for the design and engineering of novel functional peptides because hybrid peptides combine the advantages and benefits of various native peptides. In this study, to overcome all these challenges faced by the parental peptides, six hybrid peptides (CaTP, CbTP, CcTP, TPCa, TPCb, and TPCc) were designed by combining the full-length TP5 with different active fragments of CATH2. CbTP, the most potent TLR2 agonist among the six hybrid peptides, was effectively screened through in silico analysis and in vitro experiments. The CbTP peptide exhibited lower cytotoxicity than either CATH2 or TP5. Furthermore, the immunomodulatory effects of CbTP were confirmed in a CTX-immunosuppressed mouse model, which showed that CbTP has increased immunopotentiating activity and physiological stability compared to the parental peptides. CbTP successfully inhibited immunosuppression and weight loss, increased immune organ indices, and improved CD4+/CD8+ T lymphocyte subsets. In addition, CbTP significantly increased the production of the cytokine TNF-α and IL-6, and the immunoglobulins IgA, IgM, and IgG. The immunoenhancing effects of CbTP were attributed to its TLR2-binding activity, promoting the formation of the TLR2 cluster, the activation of the TLR2 receptor, and thus activation of the downstream MyD88-NF-кB signaling pathway.

Molecular ageing: free radical initiated epimerization of thymopentin--a case study.

The epimerization of amino acid residues increases with age in living organisms. In the present study, the structural consequences and thermodynamic functions of the epimerization of thymopentin (TP-5), the active site of the thymic hormone thymopoietin, were studied using molecular dynamics and density functional theory methods. The results show that free radical-initiated D-amino acid formation is energetically favoured (-130 kJmol(-1)) for each residue and induces significant changes to the peptide structure. In comparison to the wild-type (each residue in the L-configuration), the radius of gyration of the D-Asp(3) epimer of the peptide decreased by 0.5 Å, and disrupted the intramolecular hydrogen bonding of the native peptide. Beyond establishing important structural, energetic and thermodynamic benchmarks and reference data for the structure of TP-5, these results disseminate the understanding of molecular ageing, the epimerization of amino acid residues.

Influence of potential inhalation carriers on stability of thymopentin in rat bronchoalveolar lavage fluid.

In the present study, the stability of thymopentin (TP5) in bronchoalveolar lavage fluid (BALF) in presence of potential excipients in inhalation formulation was investigated. The content of TP5 was determined using HPLC method. Commonly used bulking agent, dispersibility enhancers and absorption enhancers in inhalation were investigated with respect to the stability of TP5 in BALF. Finally, the stability of TP5 in two inhalation formulations based on the screening experiments was tested in BALF. The results showed that TP5 alone degraded very rapidly in BALF and zero-order enzymatic reaction with a half-life of t0.5 = 49.20 min was observed using 10 times diluted BALF. Among the amino acids examined, leucine and phenylalanine effectively inhibited the enzymolysis of TP5 with prolonged half-life of 112.7 min and 136.2 min, respectively. Nevertheless, slight but insignificant inhibition effect was witnessed for tyrosine, aspartic acid, and lysine; and negligible prevention on the degradation process of TP5 were found for lactose and mannitol. Regarding chitosan, irrespective with molecular weight, the formation of chitosan-TP5 complex improved the stability of TP5 with prolonged t0.5 by 1.8 times. However, along with the improved stability of TP5 in spray-dried chitosan microspheres, the content of TP5 in formulations was reduced to about 75% during preparation process. Thus, leucine was proved to be a prior candidate for inhalation formulation of TP5. Consequently, the results indicate the potential of leucine as carrier for pulmonary delivery of TP5 serving as both stabilizer and dispersibility enhancer.

The in vivo immunomodulatory and synergistic anti-tumor activity of thymosin α1-thymopentin fusion peptide and its binding to TLR2.

In the present study, the immunomodulatory and synergistic anti-tumor activity of thymosin α1-thymopentin fusion peptide (Tα1-TP5) was investigated in vivo. In addition, the potential receptor of Tα1-TP5 was investigated by surface plasmon resonance (SPR) binding studies. It was found that Tα1-TP5 (305 µg/kg) alleviated immunosuppression induced by hydrocortisone (HC). Tα1-TP5 (305 µg/kg) combined with cyclophosphamide (CY) had a better tumor growth inhibitory effect than CY alone. Furthermore, Tα1-TP5 had a higher affinity (KD=6.84 µmol/L) to toll-like receptor 2 (TLR2) than Tα1 (K(D)=35.4 µmol/L), but its affinity was not significantly different from that of TP5. The results of our present work indicate that Tα1-TP5 can possibly be developed as a new immunomodulatory agent.

Intein-mediated expression, purification, and characterization of thymosin α1-thymopentin fusion peptide in Escherichia coli.

Thymosin α1-thymopentin (Tα1-TP5) fusion peptide has been proved to be an immune regulator based on its higher immunoregulatory activity than Tα1 and TP5. To obtain Tα1-TP5 more effectively and economically, Tα1-TP5 was genetically fused to a self-cleaving intein-chitin binding domain tag for purification via chitin beads in Escherichia coli. After affinity purification, the target peptide was released from the chitin beads via self-cleaving intein ((INTervening protEIN) induced by dithiothreitol. Further, Tα1-TP5 was purified by Superdex 30 and identified by Tricine-SDS-PAGE and electrospray ionization-mass spectrometry. Finally, about 7.6 mg Tα1-TP5 purified from the soluble fraction and inclusion bodies was obtained from 1 L culture media. The purity was 95% after a series of chromatographic purification steps. In vitro, the purified Tα1-TP5 could stimulate the proliferation of mouse splenic lymphocytes. Overall, this work demonstrated that Tα1-TP5 was purified with low cost and high efficiency, greatly expanding its potential use as an immune regulator.

Direct binding of thymopentin to surface class II major histocompatibility complex in living cells.

The molecular analysis of thymopentin (TP5)/class II major histocompatibility complex (MHC II) complexes has been basically understood; however, the mechanism by which TP5-MHC II complexes are formed is largely unexplored. Compared with Epstein-Barr virus (EBV)-transformed B cells expressing human leucocyte antigen DR (HLA-DR), no fluorescent signal was observed on the DR-deficient cell line. This indicates that FITC labeled TP5 (FITC-TP5) is genuinely bound to HLA-DR. The binding specificity was confirmed by incubating FITC-TP5 with unlabeled TP5 and HA peptide as well as mAb for DR molecules. In addition, the binding appeared to be rapid in living antigen-presenting cell (APC), which implies that TP5 is demonstrated on APC surface and does not require processing before associating with DR. Additional support for this surface binding arises from the observation that pretreatment of cells with a variety of metabolic inhibitors failed to decrease the level of TP5/DR complexes. However, temperature has an effect on the rate of binding between TP5 and DR molecules, which is well consistent with the qualitative predication of transition state theory. The formation of antigenic complexes is accelerated at acidic pH, which shows that the formation of TP5/DR complexes is a pH-dependent process.

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

The thymus provides a unique cellular and hormonic microenvironment for the development of immunocompetent T cells. Thymic polypeptides have been widely used clinically for the treatment of tumors, infectious diseases and immune deficiency diseases. They have already shown the ability to stimulate the maturation of hematopoietic stem cells towards the CD3+CD4+ T cell lineage. However, their effects on the thymopoiesis of embryonic stem cells are still unexplored. In this paper, we compared the effects of three thymic polypeptides, thymopentin (TP5), thymosin alpha-1 (Talpha-1) and thymopeptides on the in vitro thymopoiesis of mouse embryonic stem (ES) cells. Using the embryoid body induction system, we found that both Talpha-1 and thymopeptides effectively induced ES cells to differentiate sequentially into the CD3+ and CD4+/CD8+ T cells. These T cells had T cell receptor (TCR) Vbeta gene rearrangement and most were TCRalphabeta T cells. We also found that the expression of the Notch receptor and its ligands Delta-like-1 and Delta-like-4 gradually increased during the induction. However, TP5 failed to induce the T cell differentiation of the ES cells. In summary, this is the first report to demonstrate that Talpha-1 can stimulate the T cell early stage differentiation from ES cells using the embryoid body protocol. These findings provide a powerful model for studying T cell development and may open new venues for the clinical application of Talpha-1.

Molecular analysis of thymopentin binding to HLA-DR molecules.

Thymopentin (TP5) triggers an immune response by contacting with T cells; however the molecular basis of how TP5 achieves this process remains incompletely understood. According to the main idea of immunomodulation, we suppose that it would be necessary for TP5 to form complex with human class II major histocompatibility complex DR molecules (HLA-DR) before TP5 interacts with T cells. The uptake of TP5 by EBV-transformed B cells expressing HLA-DR molecules and the histogram of fluorescence intensities were observed by using fluorescent- labeled TP5, testifying the direct binding of TP5 to HLA-DR. The binding specificity was confirmed by the inhibition with unlabeled TP5, suggesting the recognition of TP5 by HLA-DR. To confirm the interaction between TP5 and HLA-DR, the complex formation was predicted by using various modeling strategies including six groups of trials with different parameters, alanine substitutions of TP5, and the mutants of HLA-DR. The results demonstrated that TP5 and its alanine substitutions assumed distinct conformations when they bound to HLA-DR. The observation further showed that there was flexibility in how the peptide bound within the binding cleft. Also, the molecular analysis supplemented a newly important discovery to the effect of Val anchor on TP5 binding HLA-DR, and revealed the important effects of Glu11 and Asn62 on the recognition of TP5. These results demonstrated the capability of TP5 to associate with HLA-DR in living antigen presenting cells (APC), thereby providing a new and promising strategy to understand the immunomodulation mechanism induced by TP5 and to design potential immunoregulatory polypeptides.

Increased radiation toxicity by enhanced apoptotic clearance of HL-60 cells in the presence of the pentapeptide thymopentin, which selectively binds to apoptotic cells.

Radiotoxic insult to cells is associated with genetic instability and heritable damage [Mutat. Res. 517 (2002) 173]. A strengthened response to such insult by enhanced apoptotic clearance, which would be associated with anti-inflammatory [Nature 390 (1997) 350; Nature 407 (2000) 784] and anti-necrotic intercellular signaling [Nature 418 (2002) 191], has been previously reported. The pentapeptide thymopentin (TP5) improves immunological parameters in cancer patients following radiotherapy without clinically observable side effects. We assessed the effects of TP5 on human promyeloid leukemia HL-60 cells exposed to therapeutic (2Gy) doses of X-rays. We observed an increased accumulation of cells in the G2/M phase of the cell cycle after irradiation when treated with TP5. However, TP5 had no effect on the cell cycle distribution of non-irradiated HL-60 cells. Additionally, TP5 treatment of irradiated cells increased the number of cells undergoing apoptosis. Furthermore, TP5 was found to selectively bind to apoptotic cells. These findings represent a promising and novel approach employing TP5-mediated modulation of cellular radiation response to augment both clinical gain in radiation oncology and safety measures for radiation protection.

TP5 triggers signal transduction involving mitogen activated protein kinases in monocytes.

The pentapetide thymopentin (TP5) corresponding to the aminoacids RKDVY represents the residues 32-36 of thymopoietin (TP), which was originally isolated from bovine thymus. Both were observed to induce T-cell differentiation and maturation. Recently however it was shown, that TP represents the N-terminal 49 aa of the human thymopoietin (TMPO) isoforms TMPO alpha, beta and gamma, which are localized in the nucleus. TP5 was investigated in a variety of diseases and showed efficacy by improving the immune balance, whereby different cells increased in cell number or activity. Findings which support the assumption of multifunctional efficacy and a description of TP and TP5 modulating T cells lack any interpretation on molecular level. In the present study we investigated the binding of TP5 on white blood cells. We identified monocytes and neutrophils as TP5-binding cells by displacing fluorescein-labelled TP5 with an excess of unlabelled TP5 in competition assays. Binding of TP5 on cell surface proteins resulted in cellular signalling and we report here that TP5 triggers signal transduction involving mitogen activated protein kinases p42/p44 (MAPKs) in monocytes.

CD10 (endopeptidase 24.11) is a thymic peptide-degrading enzyme possibly involved in the regulation of thymocyte functions.

Human immature thymocytes express significant levels of the CD10 (endopeptidase 24.11) cell surface antigen. We report here that IOB5, an anti-CD10 mAb, as well as the phorbol ester PMA down-regulate CD10 activity at the surface of human thymocytes. The kinetics of CD10 modulation were drastically different for both effectors, indicating different regulatory mechanisms. We also demonstrated that intact human thymocytes hydrolyze thymopentin and that CD10 significantly participates in this process. Finally, we found that thymopentin and to a lesser extent phosphoramidon, a specific endopeptidase 24.11 inhibitor, induced up-regulation of CD4 and CD8 molecules at the thymocyte cell surface. In view of these results, we suggest that down-regulation of endopeptidase 24.11 at the thymocyte cell surface might reduce its activity toward thymic factors possibly involved in the regulation of thymocyte functions.

Enzymatic cleavage of thymopoietin oligopeptides by pancreatic and intestinal brush-border enzymes.

The intestinal enzymatic degradation of the immunomodulating peptides thymotrinan (TP3), thymocartin (TP4), and thymopentin (TP5), three oligopeptides derived from the naturally occurring thymus hormone thymopoietin, was investigated to evaluate their potential for peroral drug delivery. In the presence of brush-border membrane vesicles, crude pancreas extract and everted rings from duodenum, jejunum, ileum, and colon, all peptides were shown to be degraded both by pancreatic enzymes and brush-border aminopeptidases. Degradation clearances (Cldeg) of TP3, TP4, and TP5 were calculated for a quantitative comparison of peptide stability. In the presence of crude pancreas extract, there was a rapid degradation of TP5 (Cldeg 17.9 ml/min) in comparison with TP3 and TP4 (Cldeg 0.95 and 0.56 ml/min, respectively, at 0.2 mM peptide concentration) caused by the cleavage of the C-terminal tyrosine by carboxypeptidase A, whereas TP3 and TP4 underwent hydrolysis by aminopeptidase N. In the presence of brush-border membrane vesicles, the degradation clearances were 3.9, 3.1, and 2.4 ml/min at 0.2 mM concentrations of TP4, TP5, and TP3, respectively. The clearance of all peptides was lowered with increasing peptide concentrations, indicating saturable degradation processes. The degradation of the thymopoietin oligopeptides in the presence of brush-border membrane enzymes was exclusively catalyzed by aminopeptidase N. The degradation of all peptides was highly dependent on the intestinal segment, with the lowest degradation clearance observed in the colon.