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.

[Expression and adjuvant effects of the fusion peptide TBP5].

Thymopentin (TP5) and bursopentin (BP5) are both immunopotentiators. To explore whether the TP5-BP5 fusion peptide (TBP5) has adjuvant activity or not, we cloned the TBP5 gene and confirmed that the TBP5 gene in a recombinant prokaryotic expression plasmid was successfully expressed in Escherichia coli BL21. TBP5 significantly promoted the proliferation of thymic and splenic lymphocytes of mice. The potential adjuvant activity of the TBP5 was examined in mice by coinjecting TBP5 and H9N2 avian influenza virus (AIV) inactivated vaccine. HI antibody titers, HA antibodies and cytokines levels (IL-4 and IFN-γ) were determined. We found that TBP5 markedly elevated serum HI titers and HA antibody levels, induced the secretion of both IL-4 and IFN-γ cytokines. Furthermore, virus challenge experiments confirmed that TBP5 contributed to inhibition replication of the virus [H9N2 AIV (A/chicken/Jiangsu/NJ07/05)] from mouse lungs. Altogether, these findings suggest that TBP5 may be an effective adjuvant for avian vaccine and that this study provides a reference for further research on new vaccine adjuvants.

Thymopentin nanoparticles engineered with high loading efficiency, improved pharmacokinetic properties, and enhanced immunostimulating effect using soybean phospholipid and PHBHHx polymer.

Formulation of protein and peptide drugs with sustained release properties is crucial to enhance their therapeutic effect and minimize administration frequency. In this study, immunomodulating polymeric systems were designed by manufacturing PHBHHx nanoparticles (NPs) containing thymopentin (TP5). The release profile of the drug was studied over a period of 7 days. The PHBHHx NPs containing TP5-phospholipid (PLC) complex (TP5-PLC) displayed a spherical shape with a mean size, zeta potential, and encapsulation efficiency of 238.9 nm, -32.0 mV, and 72.81%, respectively. The cytotoxicity results showed the PHBHHx NPs had a relatively low toxicity in vitro. TP5 entrapped in the NPs could hardly release in vitro, while the NPs had longer than 7 days release duration after a single subcutaneous injection in Wistar rats. The immunodepression rat model was built to evaluate the immunomodulating effects of TP5-PLC-NPs in vivo. The results of T-lymphocyte subsets (CD3(+), CD4(+), CD8(+), and CD4(+)/CD8(+) ratio) analysis and superoxide dismutase (SOD) values suggested that TP5-PLC-NPs had stronger immunoregulation effects than TP5 solution. In conclusion, an applicable approach to markedly enhancing the loading of a water-soluble peptide into a hydrophobic polymer matrix has been introduced. Thus, TP5-PLC-NPs are promising nanomedicine systems for sustained release effects of TP5.

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.

Pharmacokinetics, toxicity of nasal cilia and immunomodulating effects in Sprague-Dawley rats following intranasal delivery of thymopentin with or without absorption enhancers.

Thymopentin (TP 5), a synthetic pentapeptide, has been used in clinic as a modulator for immnuodeficiencies through intramuscular administration. The objectives of this study was to investigate the pharmacokinetics using normal rats and toxicity of nasal cilia as well as immunomodulating effects using immunosuppression rats after intranasal delivery of thymopentin with or without an absorption enhancer. The absorption extent of fluorescein isothiocyanate (FITC) labeled TP 5 via nasal delivery at a single dose is significantly improved by incorporating sodium deoxycholate, Brij 35 and chitosan, respectively. FITC-TP 5 can also be absorbed to such an extent ranging from 15 to 28% after intranasal administration of FITC-TP 5 alone, FITC-TP 5 with sodium caprylate, or with bacitracin, respectively. After seven consecutive days multiple dosing, TP 5 formulation with sodium deoxycholate or Brij 35 caused apparently injury to nasal cilia, indicating these two enhancers would not be appropriate for nasal delivery. Results from superoxide dismutase activity, maleic dialdehyde, T-lymphocyte subsets (CD3+, CD4+, CD8+ and CD4+/CD8+ ratio) analyses suggest that all the selected enhancers improve the modulating effects of TP 5 in the immunosuppression rats. On an overall evaluation, intranasal TP 5 alone, TP 5 with chitosan, or TP 5 with bacitracin formulation may be suitable for the future clinical application.

Peptides related to FKBP 39-45 loop possess immunostimulative potency.

Taking into account the sequential homology existing between thymopoietin II, the DNA-binding domain of p53 protein and FKBP (FK-506 binding protein), a series of fragments of human and bovine FKBP containing a fragment Ser39-Pro45 were synthesized. In the humoral in vitro test all the peptides act as stimulators. Whereas in the in vivo test peptides derived from bovine FKBP show an immunostimulative and those from human FKBP an immunosuppressive activity. However, after blocking the Asp residue by a Bzl group the peptide V appears to be an immunostimulator. The data obtained suggest that these peptides can influence the immune system by blocking the FKBP receptor.

Thymopentin and splenopentin as immunomodulators. Current status.

Splenopentin (SP-5, Arg-Lys-Glu-Val-Tyr) and thymopentin (TP-5, Arg-Lys-Asp-Val-Tyr) are synthetic immunomodulating peptides corresponding to the region 32-34 of a splenic product called splenin (SP) and the thymic hormone thymopoietin (TP), respectively. TP was originally isolated as a 5-kDa (49-amino acids) protein from bovine thymus while studying effects of the thymic extracts on neuromuscular transmission and was subsequently observed to affect T cell differentiation and function. TP I and II are two closely related polypeptides isolated from bovine thymus. A radioimmunoassay for TP revealed a crossreaction with a product found in spleen and lymph node. This product, named splenin, differs from TP only in position 34, aspartic acid for bovine TP and glutamic acid for bovine splenin and it was called TP III as well. Synthetic pentapeptides (TP-5) and (SP-5), reproduce the biological activities of TP and SP, respectively. It is now evident that various forms of TPs were created by proteolytic cleavage of larger proteins during isolation. cDNA clones have been isolated for three alternatively spliced mRNAs that encodes three distinct human T cell TPs. The immunomodulatory properties of TP, SP, TP-5, SP-5 and some of their synthetic analogs reported in the literature have been briefly reviewed.

Immunomodulatory activity of G-actin fragments containing a thymopentin-like sequence.

A discontinuous thymopoietin-like motif, composed of the fragments 97-111 and 277-307 of the molecule, as well as of residues Arg178 and Asn163 of G-actin was found. It was established that G-actin has an immunosuppressive activity regarding the humoral immune response. This activity is probably connected to the thymopentin-like sequence RKDLY, which is present in the 277-307 fragment of G-actin. The immunomodulatory activity of a series of peptide-partial sequences of G-actin was tested using plaque-forming cells (PFC) and delayed type hypersensitivity (DTH) tests. The investigated series consisted of five peptides: RKDLY (I), RKDLYANT (II), DVDIRKDLY (III), DVDIR (NO2)KDLY (IV), DVDIRKDLYANT (V). The peptides have the immunosuppressive activity regarding the humoral and cellular immune response.

Further investigations on thymopentin-like fragments of HLA-DQ and their analogs.

Recently we showed that the fragments of HLA-DQ with the Thr-Pro-Gln-Arg-Gly-Asp-Val-Tyr-Thr and Gln-Arg-Gly-Asp-Val-Tyr-Thr sequences strongly suppress the immune response, while their shorter analogs, Arg-Gly-Asp-Val, Arg-Gly-Asp-Val-Tyr and Gln-Arg-Gly-Asp-Val-Tyr, show very weak stimulatory activity with respect to humoral immunological response. The fragments contain the sequence which is very similar to thymopentin (pentapeptide Arg-Lys-Asp-Val-Tyr, an active fragment (32-36) of thymopoietin, an immune system activator produced in thymi), and at the same time contains the Arg-Gly-Asp (RGD) sequence, known as an inhibitor of adhesion processes. In the present study we found that a hexapeptide: Arg-Gly-Asp-Val-Tyr-Thr is the smallest size fragment of HLA-DQ having both cellular and humoral immunosuppressive activity. We also found that linear and cyclic fragments of HLA-DQ do not affect cell line production of various cytokines, what suggests that the mechanism of interactions of these peptides with the immunological system is different as compared with most other known immunosuppressors.

Immunological properties of the thymopentin-like fragments of HLA-DQ.

Class II human leukocyte antigens (HLA-II) are cell surface alpha beta heterodimers (M(r) approximately 60,000) that play a pivotal role in the immune response by presenting peptides derived from environmental antigens to the T-cell receptor. A 167-171 fragment of the beta 2-chain of the HLA-DQ molecule consists of the sequence RGDVY, which is very similar to thymopentin (pentapeptide RKDVY, an active fragment (32-36) of thymopoietin, an immune system activator produced in thymi), and at the same time contains the RGD sequence, known as an inhibitor of adhesion processes. We synthesized and investigated the immunomodulatory activity of series of peptide fragments of HLA-DQ containing thymopentin-like sequences. The results indicate that all synthesized peptides suppress the cellular immune response. However, RGDV, RGDVY and QRGDVY show very weak stimulatory activity in humoral immunological response tests. In contrast to the shorter peptides, the nonapeptide fragment of HLA-DQ, TPQRGDVYT, shows significant immunosuppressive activity in all tests. A possible role of these fragments of the polypeptide chain of HLA-DQ in the regulation of HLA functions is discussed.

The effects of immunomodulatory thymic and splenic peptides and cyclosporin A on antigen-induced arthritis in the rat.

Long-term treatment with natural and synthetic thymic and splenic peptides as well as cyclosporin A inhibited the development of antigen-induced arthritis in rats. This was demonstrated by decreased joint swelling and reduced degree of macroscopically and histologically evaluated severity of synovitis. The drug treatment also decreased serum levels of antibodies against the specific antigen methylated bovine serum albumin (mBSA) and against cartilage proteoglycans and collagens type I and II. The conclusion from these studies is that the treatment with immunomodulatory thymic and splenic peptides and with the T-cell-directed immunosuppressive drug cyclosporin A inhibits the specific immune response against mBSA and/or the development of autoimmunity against cartilage constituents. The decreased immune reactivity in the joint may reduces the severity of chronic joint inflammation.

Effect of synthetic thymic humoral factor (THF-gamma 2) on T cell activities in immunodeficient ageing mice.

Immunodeficient ageing (C57BL/10 x DBA/2)F1 mice were treated by a single injection of synthetic thymic hormones and 4 days later their thymus and spleen cells were assayed in vitro for T cell activities. A few nanograms of THF-gamma 2 were found to raise the frequency of mitogen-responsive T cells in thymus and spleen cell populations as well as the frequency of cytokine-producing splenic T cells, up to the levels observed in young mice. Moreover, injection of THF-gamma 2 was found to restore T cell growth factor (TCGF) production by mitogen-stimulated spleen cells. Also, the helper activity of spleen cells was enhanced by this treatment and increased with increasing the THF-gamma 2 dose over a wide range. Similarly, the effects of thymopentin and thymosin-alpha 1 on T helper cell activity increased with increasing the injected dose, but the efficiencies of THF-gamma 2 and thymopentin were, respectively, 400-fold and eight-fold greater than that of thymosin-alpha 1.

Systemic antibody responses to oral microorganisms in the cynomolgus monkey: development of methodology and longitudinal responses during ligature-induced disease.

Systemic antibody responses to oral microorganisms were studied during ligature-induced periodontal disease in a non-human primate (Nhp) model. Methodology was developed using ELISA techniques to assess total IgG and IgM levels in the serum from the Nhp. In addition, an ELISA was developed utilizing affinity-purified anti-human isotype reagents to detect Nhp serum antibody responses to Porphyromonas gingivalis, Actinobacillus actinomycetemcomitans, Prevotella intermedia and Fusobacterium nucleatum. Results showed that the anti-human reagents detected IgG and IgM from Macaca fascicularis with an efficiency of 25-35% and 50-60%, respectively. Following ligation, groups of Nhp were treated with an immunomodulator ("Thymopentin", TP5) or placebo to examine the effect of the T-cell stimulating agent on periodontitis and host responses. No differences were noted in total serum IgG and IgM levels for individual Nhp or between groups when baseline, ligation and treatment intervals were compared. However, following ligation, 8/12 Nhp exhibited significant increases in IgG and/or IgM antibody to P. gingivalis that were coincident with increases in the percentage of this microorganism in the subgingival plaque from the ligated sites. During the treatment phase, the antibody levels in the placebo group continued to increase, while the levels in the TP5-treated group stabilized. The findings in this study indicate that the emergence of a microorganism in the subgingival plaque (P. gingivalis) during the conversion from gingivitis to progressing periodontitis in the Nhp, elicits a systemic antibody response that is specific for the microorganism.

Reinfection of Somali children with Trichuris trichiura after chemotherapy: relevance of immunostimulation.

The intestinal helminth status of an age-stratified sample (6 to 20 years old) from a Somalian community has been assessed and the typical pattern of highly aggregated parasite distribution found. A reinfection study on a sample of 40 children (treated and untreated with a pentapeptide identical to the active site of the thymic hormone thymopoietin) seemed to indicate that immunological factors play a significant role in modulating the population dynamics of infection in endemic communities.