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Prothymosin alpha (ProTα) is a highly conserved mammalian polypeptide (109 amino acids in man) exerting in vitro and in vivo immunoenhancing activities. Recently, our team has developed a 99m Tc-radiolabeled derivative of the C-terminal bioactive decapeptide of ProTα ([ 99m Tc]C1) and employed it in in vitro studies, the results of which support the existence of binding sites on human neutrophils that recognize [ 99m Tc]C1, intact ProTα as well as the C-terminal decapeptide of ProTα and presumably involve Toll-like receptor 4. In the present work, [ 99m Tc]C1 was administered to Swiss albino mice with experimentally-induced inflammation for in vivo biodistribution and imaging studies, in parallel with a suitable negative control, which differs from [ 99m Tc]C1 only in bearing a scrambled version of the ProTα decapeptide. The biodistribution data obtained with [ 99m Tc]C1 demonstrated fast clearance of radioactivity from blood, heart, lungs, normal muscle, and predominantly urinary excretion. Most importantly, slow clearance of radioactivity from the inflammation focus was observed, resulting in a high ratio of inflamed/normal muscle tissue (9.15 at 30min post injection, which remained practically stable up to 2h). The inflammation-targeting capacity of [ 99m Tc]C1 was confirmed by imaging studies and might be attributed to neutrophils, which are recruited at the inflamed areas and bear binding sites for [ 99m Tc]C1. In this respect, apart from being a valuable tool for further studies on ProTα in in vitro and in vivo systems, [ 99m Tc]C1 merits further evaluation as a radiopharmaceutical for specific imaging of inflammation foci., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Background/objective: Prothymosin alpha (proTα) is a ubiquitous polypeptide first isolated by Haritos in 1984, whose role still remains partly elusive. We know that proTα acts both, intracellularly, as an anti-apoptotic and proliferation mediator, and extracellularly, as a biologic response modifier mediating immune responses similarly to molecules termed as "alarmins". Our research team pioneered the elucidation of the mechanisms underlying the observed activities of proTα., Results: We were the first to demonstrate that proTα levels increase during normal and abnormal cell proliferation. We showed that proTα acts pleiotropically, inducing immunomodulatory effects on immune cell populations. We revealed that the immunoreactive region of proTα is the carboxyterminal decapeptide proTα(100-109) and both molecules stimulate innate immune responses, signaling through Toll-like receptors (TLRs), specifically TLR-4. We reported that proTα and proTα(100-109) bind on the surface of human neutrophils on sites involving TLR-4, and cell activation is complemented by cytoplasmic calcium ion influx. Further, we showed that proTα and proTα(100-109) act as adjuvants upstream of lymphocyte stimulation and, in the presence of antigen, promote the expansion of antigen-reactive effectors. Most recently, we reported that proTα(100-109) may accumulate in experimentally inflamed sites and can serve as a surrogate biomarker in severe bacterial infections, proposing that extracellular release of proTα or proTα(100- 109) alerts the immune system during conditions of danger., Conclusion: We, therefore, suggest that proTα, and likely proTα(100-109), act as alarmins, being important immune mediators as well as biomarkers, and could eventually become targets for new therapeutic/diagnostic approaches in immune-related diseases like cancer, inflammation, and sepsis., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

Prothymosin alpha (ProTα) is a conserved mammalian polypeptide with intracellular functions associated with cell proliferation and apoptosis and an extracellular role associated with immunopotentiation. The N-terminal fragment [1-28], which is identical with the immunostimulating peptide thymosin α1 (Tα1), was earlier considered as the immunoactive region of the polypeptide; however, recent data suggest that ProTα may exert a discrete immunomodulating action through its central or C-terminal region, via targeting Toll-like receptor- 4 (TLR4). In this work, a derivative of the C-terminal fragment ProTα[100-109] (ProTα-D1) that can be radiolabeled with (99m)Tc was developed. The biological activity of the non-radioactive (185/187)rhenium-complex of this derivative ([(185/187)Re]ProTα-D1, structurally similar with [(99m)Tc]ProTα-D1) was verified through suitable in vitro bioassays on human neutrophils. Subsequent cell-binding studies revealed specific, time-dependent and saturable binding of [(99m)Tc]ProTα-D1 on neutrophils, which was inhibited by intact ProTα and ProTα[100-109], as well as by a "prototype" TLR4-ligand (lipopolysaccharide from Escherichia coli). Overall, our results support the existence of ProTα-binding sites on human neutrophils, recognizing [(99m)Tc]ProTα-D1, which might involve TLR4. [(99m)Tc]ProTα-D1 may be a useful tool for conducting further in vitro and in vivo studies, aiming to elucidate the extracellular mode of action of ProTα and, eventually, develop ProTα-based immunotherapeutics., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Alpha thymosins are a family of immunostimulating peptides first isolated from thymus gland. In the present work, the structure of the ReO(V)³⁺ complex of an alpha 1 thymosin derivative containing the metal-chelating N,N-dimethylglycyl-L-seryl-L-cysteinyl group was studied with NMR, CD, and ESI. The analysis indicated the existence of two interconverting diastereomers depending on the orientation of the side chain of the chelated Ser syn- or anti- to the oxygen of the ReO(V)³⁺ core. The two diastereomers could be separated on HPLC under a slow gradient showing the ratio of syn/anti to be 3:2, in agreement with the NMR data. The conversion process was shown to involve the coordination of a water molecule to the ReO(V)³⁺ core through the incubation of the complex in ¹⁸O-enriched water and subsequent ESI analysis. HPLC analysis of the analogous radioactive (⁹⁹m)TcO(V)³⁺ complex showed the formation of two isomers in the same syn/anti 3:2 ratio. Biodistribution studies of the (⁹⁹m)TcO(V)³⁺ complex in Swiss albino mice with experimentally induced inflammation showed higher accumulated radioactivity in inflamed tissue compared to normal (ratio of inflamed/control tissue 3.9). (⁹⁹m)Tc-labeled complexes of alpha thymosin derivatives are expected to facilitate research on alpha thymosins and accelerate exploitation of these peptides in immunotherapy protocols., (© 2012 John Wiley & Sons A/S.)

A derivative (1) of the immunopotentiating 28-peptide thymosin alpha1 has been especially designed, so that it can be (99m)Tc-radiolabelled, and synthesized following the Fmoc solid-phase peptide synthesis approach. Derivative 1 contains the N-terminal fragment Talpha1[1-14] as a bioactive segment, at the C-terminus of which a (99m)Tc-chelating moiety consisting of N(alpha),N(alpha)-dimethylglycine, serine and cysteine is linked through the N(epsilon)-amino group of a 'bifunctional' lysine residue; the latter is indirectly anchored on the solid-phase peptide synthesis resin through 6-aminocaproic acid (dmGSCK{N(epsilon)-Talpha1[1-14]}Aca). Synthetic derivative 1 was obtained at high overall yield (approximately 35%) and purity (>95%) and shown to be efficiently radiolabelled with (99m)Tc, thus resulting in the first, to our knowledge, so far reported (99m)Tc-radiolabelled derivative of thymosin alpha1, which may be eventually used as a specific molecular tool for the in vitro/in vivo study of the mode of action of the parent bioactive peptide.

Since conserved mammalian polypeptides are believed to exhibit enhanced immunogenicity in avian species, hens were immunized against the poorly immunogenic, highly conserved mammalian polypeptide prothymosin alpha (ProTalpha), i.e. against either non-conjugated ProTalpha (isolated from bovine thymus) or ProTalpha conjugated to keyhole limpet hemocyanin (ProTalpha/KLH). The antibodies Y were isolated from the egg yolk and evaluated through suitable dot-blot and ELISA systems in parallel with antibodies G isolated from the antiserum of rabbits immunized against the same immunogens. As revealed, antibodies Y and G of low titer and/or affinity were obtained against non-conjugated ProTalpha, while antibodies Y against ProTalpha/KLH had a better apparent titer, could better discriminate between ProTalpha and the closely related bioactive peptide thymosin alpha 1, and were obtained at much larger quantities than the corresponding antibodies G.

Alpha thymosins are a family of immunostimulating peptides first isolated from thymus gland. In the present work, the structure of the ReO(V)3+ complex of an alpha 1 thymosin derivative containing the metal-chelating N, N-dimethylglycyl- l-seryl- l-cysteinyl group was studied with NMR, CD, and ESI. The analysis indicated the existence of two interconverting diastereomers depending on the orientation of the side chain of the chelated Ser syn- or anti- to the oxygen of the ReO(V)3+ core. The two diastereomers could be separated on HPLC under a slow gradient showing the ratio of syn/ anti to be 3:2, in agreement with the NMR data. The conversion process was shown to involve the coordination of a water molecule to the ReO(V)3+ core through the incubation of the complex in 18O-enriched water and subsequent ESI analysis. HPLC analysis of the analogous radioactive 99mTcO(V)3+ complex showed the formation of two isomers in the same syn/anti 3:2 ratio. Biodistribution studies of the 99mTcO(V)3+ complex in Swiss albino mice with experimentally induced inflammation showed higher accumulated radioactivity in inflamed tissue compared to normal (ratio of inflamed/control tissue 3.9). 99mTc-labeled complexes of alpha thymosin derivatives are expected to facilitate research on alpha thymosins and accelerate exploitation of these peptides in immunotherapy protocols. [ABSTRACT FROM AUTHOR]

A derivative ( 1) of the immunopotentiating 28-peptide thymosin alpha1 has been especially designed, so that it can be 99mTc-radiolabelled, and synthesized following the Fmoc solid-phase peptide synthesis approach. Derivative 1 contains the N-terminal fragment T α1[1-14] as a bioactive segment, at the C-terminus of which a 99mTc-chelating moiety consisting of N α, N α-dimethylglycine, serine and cysteine is linked through the N ℇ-amino group of a ‘bifunctional’ lysine residue; the latter is indirectly anchored on the solid-phase peptide synthesis resin through 6-aminocaproic acid ( dmGSCK{ N ℇ-T α1[1-14]}Aca). Synthetic derivative 1 was obtained at high overall yield (approximately 35%) and purity (>95%) and shown to be efficiently radiolabelled with 99mTc, thus resulting in the first, to our knowledge, so far reported 99mTc-radiolabelled derivative of thymosin alpha1, which may be eventually used as a specific molecular tool for the in vitro/ in vivo study of the mode of action of the parent bioactive peptide. [ABSTRACT FROM AUTHOR]