促吞噬肽衍生物 T 肽对裸鼠术后残瘤 VEGF 表达的影响

目的：探讨促吞噬肽衍生物T肽抑制裸鼠术后残瘤生长的作用机理。方法：建立MCF-7乳腺癌裸鼠皮下移植瘤术后残瘤模型,观察8mg／kg剂量T肽对残余肿瘤组织的生长情况,并采用免疫组化和Western印迹检测给药后残瘤组织血管内皮生长因子（VEGF）的表达,采用RT-PCR检测不同T肽浓度对血管内皮细胞VEGF基因转录的影响。结果：T肽对MCF-7乳腺癌裸鼠皮下移植瘤术后残瘤生长表现出良好的抑制作用,按照瘤重得出的抑制率为68．2％,按照肿瘤体积得出的抑制率为67．6％,T肽给药组裸鼠残瘤组织中VEGF的表达量较空白对照组明显减少。血管内皮细胞中T肽呈剂量相关性抑制VEGF基因的转录。结论：T肽的抗癌作用与其抑制肿瘤微环境肿瘤组织和血管内皮细胞中VEGF的表达有密切联系,预示着T肽有潜力成为预防术后残瘤生长的抗癌新药。     

Synthetic peptide VKGFY and its cyclic analog stimulate macrophage bactericidal activity through non-opioid beta-endorphin receptors

We synthesized linear and cyclic pentapeptides corresponding to the sequence 369-373 of human immunoglobulin G heavy chain—VKGFY (referred to as pentarphin and cyclopentarphin, respectively). The effect of pentarphin and cyclopentarphin on phagocytosis of Salmonella typhimurium virulent 415 strain bacteria by mouse peritoneal macrophages in vitro was studied. Control experiments showed that macrophages actively captured these bacteria, but did not digest them: the captured microbes were viable and continued to proliferate inside the phagocytes; within 12 h all macrophage monolayer was destroyed (incomplete phagocytosis). If 1 nM pentarphin or cyclopentarphin was added to the cultivation medium, macrophage bactericidal activity was significantly increased and they digested all captured microorganisms within 6 h (complete phagocytosis). To study the receptor binding properties of pentarphin and cyclopentarphin we prepared ¹²⁵I-labeled pentarphin (179 Ci/mmol specific activity). The binding of ¹²⁵I-labeled pentarphin to mouse peritoneal macrophages was highaffinity (K _d = 3.6 ± 0.3 nM) and saturable. Studies on binding specificity revealed that this binding was insensitive to naloxone and [Met⁵]enkephalin, but completely inhibited by unlabeled cyclopentarphin (K _i = 2.6 ± 0.3 nM), immunorphin (K _i = 3.2 ± 0.3 nM), and -endorphin (K _i = 2.8 ± 0.2 nM). Thus, the effects of pentarphin and cyclopentarphin on macrophages are mediated by naloxone-insensitive receptors common for pentarphin, cyclopentarphin, immunorphin, and -endorphin.     

Enhancement of anti-idiotypic immune response by tuftsin in single-chain Fv-tuftsin fusion protein

Fusion protein of single-chain Fv-tuftsin (scFv-tuftsin) was constructed and expressed in yeast Pichia pastoris expression system. Immunisation of mice with scFv-tuftsin fusion protein enhanced the humoral immune responses, including anti-idiotypic (Ab2) and anti-anti-idiotypic (Ab3) inductions, when compared to scFv alone as a control. In marked contrast to response pattern in Ab2 induction, there was only a transient induction of Ab3 at day 14 of post-immunisation.     

Tuftsin signals through its receptor neuropilin‐1 via the transforming growth factor beta pathway

Tuftsin (Thr‐Lys‐Pro‐Arg) is a natural immunomodulating peptide found to stimulate phagocytosis in macrophages/microglia. Tuftsin binds to the receptor neuropilin‐1 (Nrp1) on the surface of cells. Nrp1 is a single‐pass transmembrane protein, but its intracellular C‐terminal domain is too small to signal independently. Instead, it associates with a variety of coreceptors. Despite its long history, the pathway through which tuftsin signals has not been described. To investigate this question, we employed various inhibitors to Nrp1's coreceptors to determine which route is responsible for tuftsin signaling. We use the inhibitor EG00229, which prevents tuftsin binding to Nrp1 on the surface of microglia and reverses the anti‐inflammatory M2 shift induced by tuftsin. Furthermore, we demonstrate that blockade of transforming growth factor beta (TGFβ) signaling via TβR1 disrupts the M2 shift similar to EG00229. We report that tuftsin promotes Smad3 phosphorylation and reduces Akt phosphorylation. Taken together, our data show that tuftsin signals through Nrp1 and the canonical TGFβ signaling pathway.

     

Synthesis and biological activity of tuftsin, its analogue and conjugates containing muramyl dipeptides or nor-muramyl dipeptides.

Several conjugates of muramyl dipeptide (MDP) or nor-muramyl dipeptide (nor-MDP) with tuftsin were synthesized. Conjugates 8a-f were prepared by acylation of protected tuftsin with the isoglutamine carboxyl group of MDP or nor-MDP 2a-f. Also tuftsin analogue 6 (H-Thr-Lys-Pro-Arg(NO2)-OH) was obtained. All synthesized compounds were investigated at the Medical University of Gdansk. The biological activity of the examined compounds was estimated using in vitro cultures of human monocytes and lymphocytes. The substances displayed cytotoxic effects, as was revealed in the viability tests performed. The effects were most probably mediated by the induction of an oxidative burst in monocytes and the stimulation of redox enzymes in lymphocytes. In addition, the analogues turned out to be efficient stimulators of TNFalpha and IL6 secretion by monocytes and lymphocytes. Nevertheless, the secretion of cytokines did not affect the viability of the leukocyte population used in the experiments.The beneficial properties of the compounds examined (mainly 6, 3, 8a and 8c), which implies their usefulness as potential therapeutic agents, are connected with their rapid start of action and more efficient effects compared with tuftsin alone. An in vivo assay on animal models will be performed.     

Synthesis of analogues of anthraquinones linked to tuftsin or retro-tuftsin residues as potential topoisomerase inhibitors.

A novel group of [(4-, 5- or 8)-hydroxy-9,10-anthraquinone-1-yl]-(tuftsin or retro-tuftsin) acids and methyl esters has been synthesized as potential anticancer compounds. The corresponding protected tuftsin or retro-tuftsin derivatives were also synthesized. We hope that combining compounds of different mechanisms of action will improve their clinical properties, and that our new analogues will be much more effective against multidrug-resistant tumour cell lines.     

Hydration effects on the electrostatic potential around tuftsin

The electrostatic potential and component dielectric constants from molecular dynamics (MD) trajectories of tuftsin, a tetrapeptide with the amino acid sequence Thr–Lys–Pro–Arg in water and in saline solution are presented. The results obtained from the analysis of the MD trajectories for the total electrostatic potential at points on a grid using the Ewald technique are compared with the solution to the Poisson–Boltzmann (PB) equation. The latter was solved using several sets of dielectric constant parameters. The effects of structural averaging on the PB results were also considered. Solute conformational mobility in simulations gives rise to an electrostatic potential map around the solute dominated by the solute monopole (or lowest order multipole). The detailed spatial variation of the electrostatic potential on the molecular surface brought about by the compounded effects of the distribution of water and ions close to the peptide, solvent mobility, and solute conformational mobility are not qualitatively reproducible from a reparametrization of the input solute and solvent dielectric constants to the PB equation for a single structure or for structurally averaged PB calculations. Nevertheless, by fitting the PB to the MD electrostatic potential surfaces with the dielectric constants as fitting parameters, we found that the values that give the best fit are the values calculated from the MD trajectories. Implications of using such field calculations on the design of tuftsin peptide analogues are discussed. © 1999 John Wiley & Sons, Inc. Biopoly 50: 133–143, 1999     

Cetp and exchangeable apoproteins. Common features in lipid binding activity

Tuftsin, a tetrapeptide (Thr-Lys-Pro-Arg) is known to potentiate the immunogenic activity of antigen-fed macrophages. The present study describes the mechanism of action of tuftsin in leprosy patients throughout the spectrum of the disease in vitro as a function of culture age in terms of (A) involvement of second messengers cAMP, cGMP and [Ca2+]i and (B) number of tuftsin binding sites/and their relative affinities on the monocytes/macrophages. There is apparently no direct involvement of either cAMP or cGMP while comparing the stimulated and unstimulated cultures during in vitro differentiation of monocytes (days 1, 3 and 7) or with the spectrum of the disease. Inhibition of superoxide anion release either by verapamil or with Quin 2 clearly demonstrated the involvement of [Ca2+]i as a second messenger during activation of monocytes/macrophages with tuftsin. Scatchard analysis of radiolabelled tuftsin binding data showed only one type of tuftsin receptor (low affinity) on BL/LL monoc ytes/ macrophages and normal and BT/TT cultures showed a gradual change in receptor number and affinities (low to high) with the maturation of monocytes to macrophages in contrast to BL/LL groups which displayed significantly less number of receptors. This study elicits a model which depicts that the biological responses/metabolic functions of early monocytes of normal and BT/TT gradually increase with the age of the culture till day 3 and tapers off thereafter in the older (day 7) cultures, whereas the monocytes/macrophages of BL/LL group are metabolically active only on day 1. The present study thereby implies that the clearance of leprosy bacilli from lepromatous leprosy lesions as a consequence of local or systemic immunotherapy (in the present study, the macrophage modulation by tuftsin) depends on the influx of new competent macrophages, rather than the local activation of resident lepromatous macrophages.