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.

Thymopentin ameliorates dextran sulfate sodium-induced colitis by triggering the production of IL-22 in both innate and adaptive lymphocytes.

Background: Ulcerative colitis (UC) is a chronic inflammatory gastrointestinal disease, notoriously challenging to treat. Previous studies have found a positive correlation between thymic atrophy and colitis severity. It was, therefore, worthwhile to investigate the effect of thymopentin (TP5), a synthetic pentapeptide corresponding to the active domain of the thymopoietin, on colitis. Methods: Dextran sulfate sodium (DSS)-induced colitis mice were treated with TP5 by subcutaneous injection. Body weight, colon length, colon weight, immune organ index, disease activity index (DAI) score, and the peripheral blood profile were examined. The immune cells of the spleen and colon were analyzed by flow cytometry. Histology was performed on isolated colon tissues for cytokine analysis. Bacterial DNA was extracted from mouse colonic feces to assess the intestinal microbiota. Intestinal lamina propria mononuclear cells (LPMCs), HCT116, CT26, and splenocytes were cultured and treated with TP5. Results: TP5 treatment increased the body weight and colon length, decreased the DAI score, and restored colon architecture of colitic mice. TP5 also decreased the infiltration of immune cells and expression levels of pro-inflammatory cytokines such as IL-6. Importantly, the damaged thymus and compromised lymphocytes in peripheral blood were significantly restored by TP5. Also, the production of IL-22, both in innate and adaptive lymphoid cells, was triggered by TP5. Given the critical role of IL-22 in mucosal host defense, we tested the effect of TP5 on mucus barrier and gut microbiota and found that the number of goblet cells and the level of Mucin-2 expression were restored, and the composition of the gut microbiome was normalized after TP5 treatment. The critical role of IL-22 in the protective effect of TP5 on colitis was further confirmed by administering the anti-IL-22 antibody (αIL-22), which completely abolished the effect of TP5. Furthermore, TP5 significantly increased the expression level of retinoic acid receptor-related orphan receptor γ (RORγt), a transcription factor for IL-22. Consistent with this, RORγt inhibitor abrogated the upregulation of IL-22 induced by TP5. Conclusion: TP5 exerts a protective effect on DSS-induced colitis by triggering the production of IL-22 in both innate and adaptive lymphocytes. This study delineates TP5 as an immunomodulator that may be a potential drug for the treatment of UC.

The effect of thymopentin add-on in hepatitis B e antigen positive chronic hepatitis B after virus suppression by peginterferon plus entecavir therapy.

Hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) was positively correlated with serological hepatitis B surface antigen (HBsAg) levels in hepatitis B e antigen (HBeAg) positive chronic hepatitis B (CHB) patients. We evaluated whether Thymopentin (TP5) and interferon (IFN-a) had a synergic effect on HBV cccDNA and the effect of TP5 addition therapy on HBsAg clearance in CHB patients. Real-time PCR experiments were performed to test cccDNA in HepG2.2.15 cells. 45 HBeAg-positive CHB patients had been distributed into two groups randomly. Treatment group: 23 patients were treated with a 24-week TP5 on the basis of the treatment entecavir (ETV) and peginterferon alfa-2a (PegIFN alpha-2a). Control group: 22 patients were treated with ETV and PegIFNa-2a. The study period was 72 weeks. In HepG2.2.15 cells, TP5 5ug/ml and 10ug/ml respectively combined with IFN-a 2ku/ml could potently inhibit cccDNA level at 72 hours (P<0.05). In clinical study, mean HBsAg levels in two groups are not significantly different at different time points (p=0.112). However, changes of mean HBsAg levels in TP5 add-on group at different time points are significantly different (p<0.05). Patients with HBsAg levels <1500IU/ml in control group had higher HBsAg levels compared with patients with HBsAg levels <1500IU/ml in TP5 add-on group (P=0.019). The latter had the most pronounced HBsAg reduction. TP5 and IFN had a synergic effect on inhibiting cccDNA levels in HepG2.2.15 cells; Patients in treatment group showed no extra side effects compared with the control group. 24 weeks TP5 add-on treatment was safe and had a tendency to accelerate the decline of HBsAg when HBV-DNA was undetectable.

Thymopentin enhances the generation of T-cell lineage derived from human embryonic stem cells in vitro.

Thymopentin is a group of biologically active peptide secreted mainly by the epithelial cells of thymic cortex and medulla. Whether it promotes T cells production from human embryonic stem cells(hESCs) in vitro remains an elusive issue. In the present study, we develop a novel strategy that enhances T-cell lineage differentiation of hESCs in collagen matrix culture by sequential cytokine cocktails treatment combined with thymopentin stimulation. We observed that approximately 30.75% cells expressed CD34 on day 14 of the cultures and expressed the surface markers of erythroid, lymphoid and myeloid lineages. The results of colony assays and gene expressions by RT-PCR analysis also demonstrated that hematopoietic progenitor cells (HPCs) derived from hESCs were capable of multi-lineage differentiation. Further study revealed that culturing with thymopentin treatment, the CD34(+)CD45RA(+)CD7(+) cells sorted from HPCs expressed T-cell-related genes, IKAROS, DNTT, TCRγ and TCRß, and T-cell surface markers, CD3, cytoplasmic CD3, CD5, CD27, TCRγδ, CD4 and CD8. The differentiated cells produced the cytokines including IFN-γ, IL-2 and TNF-α in response to stimulation, providing the evidence for T-cell function of these cells. In conclusion, thymopentin enhances T-cell lineage differentiation from hESCs in vitro by mimicking thymus peptide environment in vivo.

The novel role of thymopentin in induction of maturation of bone marrow dendritic cells (BMDCs).

Thymopentin is an immune-modulating peptide that can stimulate cellular immune responses and has been used in many immune handicapped cases [1]. However, despite documented reports proving its efficacy in immunoregulation, there have been no reports, as yet, concerning its impact on the maturation and function of dendritic cells (DCs). In this study, we analyzed the effects of thymopentin on the detailed regulation of maturation of murine bone-marrow-derived DCs (BMDCs). The phenotypic and structural maturation of BMDCs was confirmed by transmission electron microscopy (TEM) and flow cytometry (FCM). The functional maturation was confirmed by an acid phosphatase (ACP) activity test, FITC-dextran bio-assay, test of 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE), labeled CD4(+)T cell proliferation and enzyme-linked immunosorbent assay (ELISA). We determined that thymopentin up-regulated the expression of CD40, CD80, CD86, CD83, and MHC II molecules on BMDCs, down-regulated phagocytosis of BMDCs, increased BMDCs driven CD4(+)T cell proliferation, and enhanced BMDC production of IL-12 and TNF-α. Therefore, we concluded that thymopentin highly induces BMDC maturation and intensifies DC/T-cell pathways. These data also provide direct evidence and rationale concerning the potential clinical use of thymopentin in various immune handicapped cases and suggest that thymopentin should be considered as a potent adjuvant for DC-based vaccines.

Synthetic modifications of the immunomodulating peptide thymopentin to confer anti-mycobacterial activity.

Effective global control of tuberculosis (TB) is increasingly threatened by the convergence of multidrug-resistant TB and the human immunodeficiency virus (HIV) infection. TB/HIV coinfections exert a tremendous burden on the host's immune system, and this has prompted the clinical use of immunomodulators to enhance host defences as an alternative therapeutic strategy. In this study, we modified the clinically used synthetic immunomodulatory pentapeptide, thymopentin (TP-5, RKDVY), with six arginine residues (RR-6, RRRRRR) at the N- and C-termini to obtain the cationic peptides, RR-11 (RKDVYRRRRRR-NH2) and RY-11 (RRRRRRRKDVY-NH2), respectively. The arginine residues conferred anti-mycobacterial activity to TP-5 in the peptides as shown by effective minimum inhibitory concentrations of 125 mg/L and killing efficiencies of >99.99% against both rifampicin-susceptible and -resistant Mycobacterium smegmatis. The immunomodulatory action of the peptides remained unaffected as shown by their ability to stimulate TNF-α production in RAW 264.7 mouse macrophage cells. A distinct change in surface morphology after peptide treatment was observed in scanning electron micrographs, while confocal microscopy and dye leakage studies suggested bacterial membrane disruption by the modified peptides. The modified peptides were non-toxic and did not cause hemolysis of rat red blood cells up to a concentration of 2000 mg/L. Moreover, RY-11 showed synergism with rifampicin and reduced the effective concentration of rifampicin, while preventing the induction of rifampicin resistance. The synthetic peptides may have a potential application in both immunocompetent and immunocompromised TB patients.

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.

Repeated transfusions of autologous cytokine-induced killer cells for treatment of haematological malignancies in elderly patients: a pilot clinical trial.

The elderly population is susceptible to haematological malignancies, and these elderly patients are intolerant to cytotoxic drugs. Therefore, the exploration of a safe and reliable strategy exclusive of chemotherapy is critical in improving the prognosis of elderly patients with haematological malignancies. We evaluated the safety and the efficacy of autologous cytokine-induced killer (CIK) cells combined with recombinant human interleukin 2 (rhIL-2) in the treatment of haematological malignancies in elderly patients. Peripheral blood mononuclear cells were isolated from 20 elderly patients with haematological malignancies, then augmented by priming with interferon gamma, rhIL-2 and CD3 monoclonal antibody. The autologous CIK cells (2-3 × 10(9)) were transfused back to patients, followed by a subcutaneous injection of IL-2 (1 mU/day) for 10 consecutive days. The regimen was repeated every 4 weeks. The host cellular immune function, tumour-related biological parameters, imaging characteristics, disease condition, quality of life and survival time were assessed. Fourteen patients received 8 cycles of transfusion and 6 received 4 cycles. No adverse effects were observed. The percentages of CD3(+), CD3(+) CD8(+) and CD3(+) CD56(+) cells were significantly increased (p < 0.05), and the levels of serum ß2 microglobulin and lactate dehydrogenase (LDH) were markedly decreased (p < 0.05) after autologous CIK cell transfusion. Cancer-related symptoms were profoundly alleviated, as demonstrated by the improved quality of life (p < 0.01). Complete remission was observed in 11 patients, persistent partial remission in 7 patients and stable disease in 2 patients. At the end of follow-up, the mean survival time was 20 months. Transfusion with autologous CIK cells plus rhIL-2 treatment is safe and effective for treating haematological malignancies in elderly patients.

Multivesicular liposomes for the sustained release of thymopentin: stability, pharmacokinetics and pharmacodynamics.

The objective of the present study was to investigate the storage stability of thymopentin multivesicular liposomes (TP5-MVLs) prepared with different emulsifiers, and to study the pharmacokinetics and pharmacodynamics of the produced TP5-MVLs in vivo. The stability studies of TP5-MVLs indicated that MVLs particles prepared with mixed emulsifiers (Myrj52:solutolHS15 = 2:3) were stable at the storage temperature of 4 +/- 2 degrees C within 3 months. In addition, FITC-TP5-loaded MVLs was prepared for pharmacokinetic studies that after subcutaneous administration, the fluorescence signal lasted for about 5 days in plasma demonstrating that the rate of drug release from MVLs was very slow. The pharmacodynamic studies indicated that the therapeutic efficacy of TP5-MVLs after subcutaneous administration once every four days was the same as free TP5 solution after intravenous or subcutaneous administration once daily. In conclusion, MVLs, which possessed great storage stability, can be utilized to reduce the administration frequency of TP5, and therefore, served as a promising sustained release delivery system for polypeptide.

Thymopentin down-regulates both activity and expression of iNOS in blood cells of Sézary syndrome patients.

While thymopentin has been used for many years in the experimental treatment of Sézary syndrome (SS), a rare and very aggressive lymphoma, its mechanism of action is still not known. Herein we show that this peptide acts as an inhibitor of isolated iNOS and nNOS isoforms, and reduces iNOS protein/mRNA levels and iNOS activity in blood cells obtained from both healthy donors and SS patients. Similar results were obtained with TPN-2, the N(ω)-nitro analogue of the Arg-Lys motif present in thymopentin. Additional investigations are necessary to confirm the role and the relative importance of the two mechanisms of iNOS down-regulation in the therapeutic action of these peptides against SS.

Thymus peptides regulate activity of RAW 264.7 macrophage cells: inhibitory analysis and a role of signal cascades.

OBJECTIVES: The aim of this study was to reveal T-lymphocyte-independent mechanisms of thymic peptide-mediated immunomodulation. METHODS: The effects of two thymic peptides- thymulin and thymopentin were studied in cultured RAW 264.7 macrophages (lipopolysaccharide-stimulated or unstimulated) by measuring cytokine production and signal protein levels. RESULTS: Both peptides increased proinflammatory cytokine secretion by unstimulated RAW 264.7 macrophages and these effects were blocked by the NF-κB cascade inhibitor, stress-activated protein kinase (SAPK)/JNK cascade inhibitor and, to a lesser extent, Toll-like 4 receptor activity inhibitor. In macrophages stimulated by bacterial lipopolysaccharide, peptides alone did not affect cytokine secretion, but significantly enhanced effects of each of the inhibitors. Thymopentin increased activation of both NF-κB and SAPK/JNK cascades in unstimulated macrophages, while thymulin significantly decreased activation of the SAPK/JNK but not NF-κB cascade in LPS-stimulated macrophages. Thymulin and thymopentin increased production of the heat shock protein HSP72 both in LPS-stimulated and unstimulated cells. CONCLUSIONS: Thymulin and thymopentin are effective anti-inflammatory modulators with direct actions on innate immune cells; the effects involve multiple signal cascades, including NF-κB and SAPK/JNK pathways. Since signaling cascades are now considered to be targets for new therapies, thymic peptides may be prospective modulators of signaling cascades, acting alone or in combination with other agents.

Expression of CD34 in hematopoietic cancer cell lines reflects tightly regulated stem/progenitor-like state.

Hematopoietic cancer stem cells preserve cellular hierarchy in a manner similar to normal stem cells, yet the underlying regulatory mechanisms are poorly understood. It is known that both normal and malignant stem/progenitor cells express CD34. Here, we demonstrate that several cell lines (HL-60, U266) derived from hematopoietic malignancies contain not only CD34(-) but also CD34(+) subpopulations. The CD34(+) cells displayed a stem/progenitor-like phenotype since, in contrast to CD34(-) cells, they frequently underwent cellular division and rapidly formed colonies in methylcellulose-based medium. Strikingly, a constant fraction of the CD34(+) and CD34(-) cell subpopulations, when separated, rapidly switched their phenotype. Consequently, both separated fractions could generate tumors in immunocompromised NOD/LtSz-scid/scid mice. Cultures in vitro showed that the proportion of CD34(+) stem/progenitor-like cells in the population was decreased by cell-cell contact and increased by soluble factors secreted by the cells. Using cytokine arrays, we identified some of these factors, notably thymopoietin that was able to increase the proportion of CD34(+) cells and overall colony-forming capacity in tested cell lines. This action of thymopoietin was conserved in mononuclear cells from bone marrow. Therefore, we propose that hematopoietic cancer cell lines containing subpopulations of CD34(+) cells can provide an in vitro model for studies of cancer stem/progenitor cells.

[Immunomodulatory effects of thymopentin under acute and chronic inflammations in mice].

The effects of thymopentin, a synthetic analogue of the active center of the thymus hormone thymopoietin, on the immune status of mice with two different models of inflammation induced by injection of lipopolysaccaride (LPS) from gram-negative bacteria were studied. Acute inflammation was induced by a single injection of LPS in a dose of 250 microg/100 g of body weight, and chronic inflammation (sepsis) was modeled by a daily injection of LPS for 11 days with a gradual increase in the dose range from 25 to 250 microg/100 g of body weight. Under acute inflammation, a preliminary injection of thymopentin did not induce any additional stimulation in cytokine production increased by LPS. On the contrary, whereas the chronic introduction of LPS was characterized by a depressed production of several cytokines, thymopentin produced an immunostimulating effect. Thus, an increase in the production of nitric oxide, interferon-gamma, and Hsp70 was demonstrated. In addition, a more effective restoration of the number of thymus cells, as well as an increase in macrophage tumor necrosis factor-alpha production were observed after repeal of LPS + hormone injections. The results show that preliminary application of thymopentin promotes the regulation of immune cell activity under acute and chronic inflammation.

[Mediation of cellular immune response by TP5 in pathogenesis of myasthenia gravis].

OBJECTIVE: To evaluate the effects of TP5 upon the production of IFN-gamma and different T cell subsets by human peripheral blood mononuclear cells (PBMCs) from patients with myasthenia gravis (MG) and to provide experimental rationales for TP5 in clinical therapy of MG. METHODS: PBMCs were isolated from peripheral blood of MG individuals and cultured with anti-CD3. The level of IFN-gamma in culture supernatants was examined by ELISA. The subsets and frequency of IFN-gamma-producing cells were examined at a single-cell level by flow cytometry. RESULTS: After PBMCs stimulation with anti-CD3 and TP5 (300 microg/ml), the level of IFN-gamma expression was significantly inhibited (P(child) = 0.0001, P(adult) = 0.01); and the level of IFN-gamma expression from normal adult and child controls was also significantly inhibited (P(child) = 0.009, P(adult) = 0.0001). In addition, the inhibition of TP5 on the production of IFN-gamma by PBMCs from MG children was lower compared with normal child control. But as compared with normal adult control, the inhibition of TP5 showed no significant difference in MG adults (P(adult) = 0.481). TP5 inhibited the expression of IFN-gamma by CD8+ T cell and CD4+ T cell. CONCLUSION: TP5 can inhibit the response of cellular immune by decreasing the production of IFN-gamma in MG consequence display that the level of IFN-gamma significant decreased with the addition of TP5 and anti-CD3. But after considering the age, the level of IFN-gamma in MG children was no as much inhibited as normal child. TP5 inhibits the expression of IFN-gamma by CD8+ T and CD4+ T cells.

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

Thymopentin plays an important role in improving imbalanced immune systems of patients, however, it has a limited half-life in plasma. To get more stable and active thymopentin analogs, a fusion thymosin alpha1-thymopentin (Talpha1-TP5) gene was synthesized and cloned into vector pGAPZalphaA. Talpha1-TP5 fusion peptide was expressed in pichia pastoris and purified by metal chelating chromatography and gel filtration chromatography. The circular dichroism spectra (CD) indicated that the secondary structure of Talpha1-TP5 fusion peptide is dominated by a-helix and random coil. In vitro analysis showed that the plasma half-life of Talpha1-TP5 fusion peptide is 140 +/- 14 min, which is longer than that of TP5 (5.6+/-0.7 min) and Talpha1 (127+/-11 min). The in vitro activity assay presented that Talpha1-TP5 fusion peptide has greater activity in promoting proliferation of Kunming mouse splenocytes, and in vivo experiment it showed better activity in promoting the phagocytosis of macrophages and secretion of IL-2 than both Talpha1 and TP5. Our findings suggest that Talpha1-TP5 fusion peptide might be a potential therapeutic agent.

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.

Conjugation of thymopentin (TP5) with lipoamino acid residues increases the hydrolytic stability and preserves the biological activity.

Three conjugates of thymopentin (TP5), an oligopeptide derived from the thymic hormone thymopoietin, with lipoamino acid (LAAs) have been obtained by solid-phase peptide synthesis. Both linear and dendrimer structures have been prepared to achieve enhanced lipophilicity. After incubation in foetal calf serum the lipophilic conjugates showed a higher stability to hydrolysis with respect to the parent drug. In a preliminary in vitro biological assay, LAA conjugates showed the ability to retain or improve the growth inhibitory activity of the parent peptide against a human lymphoblastoid cell line. The interaction of the prepared conjugates with 1,2-L-alpha-dimiristoylphosphatidylcholine multilamellar liposomes, chosen as a biological membrane model, was studied. The higher lipophilicity of TP5 conjugates was reflected in a better penetration through phospholipid bilayers, whose thermal behaviour was altered in a concentration-dependent way. Such enhanced affinity of TP5-LAA conjugates for this membrane model could anticipate a better interaction with cell membranes and, ultimately, an improved biological activity of compounds compared with the parent pentapeptide.

Chemical synthesis and application of C-terminally 5-carboxyfluorescein-labelled thymopentin as a fluorescent probe for thymopoietin receptor.

Thymopentin (TP5) is a synthetic pentapeptide fragment, which corresponds to position 32 - 36 of thymic polypeptide thymopoietin. Thymopoietin and TP5 display a variety of biological functions, including phenotypic differentiation of T cells and the regulation of immune systems. Previous chemical modification experiments suggested that there was an absolute requirement for N-terminal amino acids to maintain the biological activity of TP5. On the basis of this structure-activity relationship, we designed and synthesized the C-terminally 5-carboxyfluorescein-coupled TP5 (TP5-FAM) as a fluorescent probe for thymopoietin receptor. TP5-FAM could bind to the membrane of human lymphoid cell lines, MOLT-4 cells, in which the thymopoietin receptor is expressed. The binding is specific and saturable (K(d) = 33 microM). TP5 and human splenopentin are nearly equipotent inhibitors of TP5-FAM binding to the thymopoietin receptor, but porcine secretin did not show any significant inhibition of TP5-FAM binding to MOLT-4 cells. Thus, TP5-FAM is suggested to be a potent and biologically active ligand that would be useful for studying the binding and functional characteristics of the human thymopoietin receptor.

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

Thymopentin (Arg-Lys-Asp-Val-Tyr, TP5) has shown immuno-regulatory activities in humans. In the present study, we investigated the effects of TP5 on the proliferation and differentiation of a human promyelocyte leukemia cell line, HL-60. It is noteworthy that TP5 displayed concentration-dependent inhibitory effects on the proliferation and colony formation of HL-60 cells. Furthermore, the decrease or even disappearance of AgNORs from nucleoli was observed in HL-60 cells after the treatment with TP5. The suppression induced by TP5 was accompanied by an accumulation of cell cycle in the G0/G1 phase. Moreover, TP5 significantly increased the NBT-reduction activity of HL-60 cells. Cytofluorometric and morphologic analysis indicated that TP5 had induced differentiation along the granulocytes lineage in HL-60 cells. d-tubocurarine (TUB) significantly antagonized the inhibitory effects induced by TP5, whereas atropine did not exhibit such effect. All the results indicated that TP5 was able to significantly inhibit proliferation and induce differentiation in HL-60 cells. Our observations also implied that TP5 not only acted as an immunomodulatory factor in cancer chemotherapy, but is also a potential chemotherapeutic agent in the human leukemia therapy.

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.