Pharmacological inhibition of p38 potentiates antimicrobial peptide TP4-induced cell death in glioblastoma cells.

Glioblastoma is the most common and deadly type of brain cancer. The poor prognosis may be largely attributed to inadequate disease response to current chemotherapeutic agents. Activation of p38 is associated with deleterious outcomes in glioblastoma patients, as its signaling mediates chemoresistance mechanisms. Antimicrobial peptide tilapia piscidin (TP) 4 was identified from Nile tilapia (Oreochromis niloticus) and exhibits strong bactericidal effects on Gram-positive and Gram-negative bacteria. TP4 also has anticancer activity toward human triple-negative breast cancer cells and glioblastoma cells. In the present study, we tested the cytotoxic effects of combined TP4 and p38 inhibitors on glioblastoma U251 cells. We found that the combination of TP4 and p38 inhibitors (SB202190 and VX-745) enhanced cytotoxicity in U251 glioblastoma cells but not noncancerous neural cells. Cytotoxicity from the combination treatments proceeded via necrosis and not apoptosis. Mechanistically, SB202190 potentiated TP4-induced mitochondrial dysfunction, reactive oxygen species generation and unbalanced antioxidant status, which resulted in necrotic cell death. Thus, we demonstrated for the first time that combinations of TP4 and p38 inhibitors have the potential to preferentially target glioblastoma cells, while sparing noncancerous neural cells.

Efficacy of the antimicrobial peptide TP4 against Helicobacter pylori infection: in vitro membrane perturbation via micellization and in vivo suppression of host immune responses in a mouse model.

Helicobacter pylori infection is marked by a strong association with various gastric diseases, including gastritis, ulcers, and gastric cancer. Antibiotic treatment regimens have low success rates due to the rapid occurrence of resistant H. pylori strains, necessitating the development of novel anti-H. pylori strategies. Here, we investigated the therapeutic potential of a novel peptide, Tilapia Piscidin 4 (TP4), against multidrug resistant gastric pathogen H. pylori, based on its in vitro and in vivo efficacy.TP4 inhibited the growth of both antibiotic-sensitive and -resistant H. pylori (CagA+, VacA+) via membrane micelle formation, which led to membrane depolarization and extravasation of cellular constituents. During colonization of gastric tissue, H. pylori infection maintains high T regulatory subsets and a low Th17/Treg ratio, and results in expression of both pro- and anti-inflammatory cytokines. Treatment with TP4 suppressed Treg subset populations and pro- and anti- inflammatory cytokines. TP4 restored the Th17/Treg balance, which resulted in early clearance of H. pylori density and recovery of gastric morphology. Toxicity studies demonstrated that TP4 treatment has no adverse effects in mice or rabbits. The results of this study indicate that TP4 may be an effective and safe monotherapeutic agent for the treatment of multidrug resistant H. pylori infections.

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.

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.

Inhibition of thymidine phosphorylase (PD-ECGF) from SD-lymphoma by phosphonomethoxyalkyl thymines.

A series of thymine phosphonomethoxyalkyl derivatives were evaluated for their ability to inhibit thymidine phosphorylase (dThdPase) purified from rat spontaneous T-cell lymphoma. A kinetic study of thymidine phosphorolysis catalyzed by dThdPase was performed with thymidine and/or inorganic phosphate as substrates. Data show that the substantial inhibitory effect of these acyclic nucleotide analogues is decreasing in the order of (R)-FPMPT>(S)-FPMPT>or=(R)-HPMPT>(S)-PMPT>(S)-HPMPT>PMET>or=(R)-PMPT. The inhibitory potency (K(i)/(dThd)K(m)) of the most efficient inhibitors from this series against T-cell lymphoma enzyme is 0.0026 for (R)-FPMPT and 0.0048 for (S)-FPMPT. The studied compounds do not inhibit Escherichia coli and human enzyme and possess lower inhibitory potency against rat liver thymidine phosphorylase.

[Correction of immunodeficiency status caused by herbicide 2,4-D in experiment]. / Korrektsiia immunodefitsitnogo sostoianiia, vyzvannogo gerbitsidom 2,4-D v éksperimente.

A possibility to correct the immunodeficiency state, caused by herbicide 2.4-D (20 mg/kg of weight daily during 5 days), by using immunomodulators Levamisole (perorally), Tactivinum (hypodermically) and Spleninum (hepodermically) in reactions Graft-versus-Host (GH), Antibody-Forming Cells (AFG) and phagocytic activity of peritoneal macrophages in vivo and in vitro etc, was studied in experiments with 360 mice of lines CBA and F1 (CBAxC57B1/6) weighing 18 to 22 g. It was established that Levamisole (2.5 mg/kg per body weight, for 1 to 3 days) and Tactivinum (0.2 mkg/mouse, for 1 to 3 days) had an immunocorrecting effect in poisoning by 2.4-D. Spleninum (10 mcl/mouse) corrected mainly the humoral chain in the immune response with the AFG recovery to the level observed in intact animals (controls). The data on the influence produced by the immunomodulators on the phagocytic activity in vitro correlated with such data obtained in vivo.

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.

Upregulation of HLA class-I gene transcription in K562 cells by analogs of splenopentin (SP-5)

We report here the capacity of three analogs of Splenopentin, to up regulate transcription of the HLA-B7 gene in the K562 cell line. These cells normally do not transcribe the HLA class I genes. The Splenopentin analogs used in this study are effective at very low molar concentrations and are non-toxic to cells at these levels. Electrophoretic Mobility Shift assays indicate that this transcriptional up regulation of HLA class I genes may be related to the appearance of novel class I promoter binding factors induced in the nuclei of treated cells.

Solid-phase peptide synthesis and biological activity of bovine thymopoietin II (bTP-II).

Bovine thymopoietin (bTP), a 49 amino acid polypeptide, was synthesized using Merrifield's solid-phase peptide synthesis methodology. The polypeptide was purified using anion-exchange chromatography and reversed-phase HPLC and characterized by mass spectrometry and amino acid analysis of the full-length peptide and of products derived from digestion with Staphylococcus aureus V8 protease. The biological activity of the synthesized product was tested in several assay systems. Synthetic bTP was found to induce the expression of Thy 1.2 antigen on T-lymphocytes from athymic mice, in agreement with previous studies on the biological activity of endogenous bTP. Biological activity at skeletal muscle and neuronal nicotinic acetylcholine receptor sites, as reported by others for bTP, could not be confirmed in our studies. The absence of biological activity at nicotinic receptor sites may be related to the results of a recent report demonstrating the presence of a cobratoxin-like molecule in preparations of natural bTP. These data indicate that synthetic peptides have an important role for the evaluation of the specificity of the biological activity of polypeptides.

The effect of splenopentin (DA SP-5) on in vitro myelopoiesis and on AZT-induced bone marrow toxicity.

Splenopentin (DA SP-5) is a pentapeptide corresponding to the amino acid sequence 32-36 (Arg-Lys-Glu-Val-Tyr) of the splenic hormone splenin. We examined the influence of DA SP-5 on bone marrow progenitor cell (BMC) proliferation. DA SP-5 acts as a co-stimulant for recombinant human granulocyte-macrophage colony-stimulating factor (rHuGM-CSF) in the induction of human BMC derived colony formation in vitro (colony-forming units). When exposed to DA SP-5 and thereafter to AZT and rHuGM-CSF, BMCs show a colony-forming response similar to that after cultivation with the rHuGM-CSF alone. In contrast, when exposed to AZT and rHuGM-CSF (and not preincubated with DA SP-5) the colony formation was reduced. A similar pentapeptide thymopentin (Arg-Lys-Asp-Val-Tyr) did not influence colony formation by human BMCs. We assume that DA SP-5 could support therapeutic effects of rHuGM-CSF.

Thymopoietin, a thymic polypeptide, prevents nicotinic agonist-induced morphological changes in neonatal muscle cells in culture.

Thymopoietin, a polypeptide hormone isolated from thymus and involved in immune function, potently inhibited [125I]alpha-bungarotoxin binding to neonatal muscle cells in culture (IC50 = 3.8 nM) and blocked carbachol-stimulated 22Na uptake with an IC50 of 1.9 +/- 0.2 nM and 23 +/- 7 nM at a half-maximal and maximal concentration of carbachol, respectively. Studies were subsequently done to evaluate potential long-term functional consequences of this interaction of thymopoietin at the nicotinic receptor. Exposure (1-3 days) of neonatal muscle cells in culture to nicotine (3 x 10(-6) M) or carbachol (1 x 10(-6) M) resulted in a decline in myotube branching and a decrease in myotube length. Thymopoietin did not appreciably alter myotube morphology on its own; however, it prevented the effects of nicotine and carbachol on muscle cell morphology at concentrations (1-10 nM) which corresponded well to those with which thymopoietin interacted at the receptor. The action of alpha-bungarotoxin on the myotubes was very similar to that of thymopoietin. These studies suggest that the endogenously occurring polypeptide, thymopoietin, has the potential to modulate muscle cell morphology through an interaction at the nicotinic receptor.

Combined effects of a thymic peptide, thymopoietin and myasthenic patient sera in rat myotube culture.

We investigated in a rat myotube assay the combined effect of 26 myasthenic (MG) patient sera and a thymic peptide, thymopoietin (Tpo) which had previously been shown to bind Torpedo and human AChR and to compete with alpha-bungarotoxin (alpha-Bgt) binding. Cultures were first exposed to Tpo alone for 3 h (0.3, 7.5, 15 nM), then MG sera (5% final dilution) were added for an additional 18 h. Reduction in the amount of 125I-alpha-Bgt binding sites in the presence of various concentrations of Tpo were similar with control sera and in all the patients with low or undetectable anti-AChR Ab (11 cases). In cultures exposed to Tpo and sera with high anti-AChR Ab titre (15 cases), Tpo and anti-AChR Ab have an additive capacity to reduce the number of alpha-Bgt binding sites. The results are compatible with the hypothesis that anti-AChR Ab and Tpo could impair neuromuscular transmission by complementary mechanisms.

Thymopoietin, a polypeptide ligand for the alpha-bungarotoxin binding site in brain: an autoradiographic study.

Thymopoietin, a 48-49-amino acid polypeptide present in the thymus gland, was investigated as a potential ligand for the neuronal nicotinic alpha-bungarotoxin binding site in rat brain. Binding of [125I]alpha-bungarotoxin to whole rat brain sections was inhibited by thymopoietin in a concentration-dependent manner with an IC50 of 30.0 +/- 8.2 nM as compared to 1.1 +/- 0.3 nM for alpha-bungarotoxin. However, at concentrations of thymopoietin of up to 1 microM, [3H]nicotine binding to high affinity sites was not inhibited. Thysplenin, a polypeptide with considerable homology to thymopoietin did not affect [125I]alpha-bungarotoxin binding. These results suggest that thymopoietin selectively interacts with the nicotinic alpha-bungarotoxin binding site labelled by [125I]alpha-bungarotoxin rather than the neuronal nicotinic receptor(s) labelled by [3H]nicotine. Autoradiographic studies revealed that 1 microM thymopoietin almost completely inhibited [125I]alpha-bungarotoxin binding in all brain regions. Computer-assisted image analysis of displacement curves was performed on various brain areas rich in alpha-bungarotoxin binding, such as the dorsal endopiriform nucleus, fields 1 and 2 of Ammon's horn, the polymorph cell layer of the dentate gyrus and cortical layers 4 and 5. Thymopoietin inhibited [125I]alpha-bungarotoxin binding with similar potency in all these regions, suggesting that it interacted at the same site in the different brain areas. The IC50 values averaged over the six regions were 24.6 +/- 2.8 nM for thymopoietin and 1.2 +/- 0.2 nM for alpha-bungarotoxin. These results show that thymopoietin specifically interacted with the alpha-bungarotoxin site with a similar potency in different brain regions. It is suggested that thymopoietin represents a selective ligand for alpha-bungarotoxin binding sites in brain.

Thymopoietin, a potent antagonist at nicotinic receptors in C2 muscle cell cultures.

Recent work has shown that thymopoietin, a polypeptide with actions in the immune and nervous systems, potently binds to the alpha-bungarotoxin (alpha-BGT) receptor. The present study was done to characterize the interaction of thymopoietin at the nicotinic alpha-BGT binding site in cultured muscle cells and to correlate these findings with the effects of the polypeptide on nicotinic receptor-mediated function. Inhibition studies showed that thymopoietin potently inhibited 125I-alpha-BGT binding in C2 muscle cells in culture, with an IC50 of 1.1 nM, a value similar to that for alpha-BGT. Thymopoietin bound to the alpha-BGT receptor in the cells in culture relatively slowly; at 10(-8) M thymopoietin, maximal inhibition occurred after 45 to 75 min of exposure to the polypeptide. Dissociation of thymopoietin from the receptor exhibited a much longer time course; recovery of alpha-BGT binding to control values after exposure to 10(-8) M thymopoietin occurred approximately 16 hr after removal of the polypeptide. The effects of thymopoietin on 125I-alpha-BGT binding correlated well with those on nicotinic function. Thymopoietin potently inhibited nicotinic receptor-mediated 22Na uptake in muscle cells in culture, with an IC50 of 2 nM. This effect was dependent on the length of the preincubation period with thymopoietin, with maximal inhibition occurring after 60 min of exposure to the polypeptide. Recovery of the functional response after thymopoietin (10(-8) M) exposure required about 16 hr. The mode of inhibition of receptor-mediated ion flux by thymopoietin was similar to that observed with alpha-BGT but distinct from that obtained with d-tubocurarine and gallamine. To conclude, thymopoietin, a thymic polypeptide associated with the immune system, potently inhibited both 125I-alpha-BGT binding and nicotinic receptor-mediated function in C2 muscle cells. These findings may have implications for myasthenia gravis and/or other neuromuscular disorders.

A modified E-rosette assay as a semi-empirical tool in search of new T lymphocyte stimulants.

An "activity index" is defined for T cell modulators, which allows meaningful comparison of experimental results by eliminating the deviations due to differences in the immunological states of T lymphocytes.

Interactions of the thymic polypeptide hormone thymopoietin with neuronal nicotinic alpha-bungarotoxin binding sites and with muscle-type, but not ganglia-type, nicotinic acetylcholine receptor ligand-gated ion channels.

Studies were conducted to assess the ability of the thymic polypeptide hormone thymopoietin (TPO) to interact with proto-typical ganglia-type or muscle-type nicotinic acetylcholine receptor ion channels (nAcChoR). Also investigated were interactions of TPO with neuronal nicotinic alpha-bungarotoxin binding sites (nBgtS), which share many features with nAcChoR and may belong to an extended nAcChoR family but do not appear to function as simple ligand-gated ion channels. TPO and alpha-bungarotoxin (Bgt) share the capacity for high affinity (IC50 values in the nanomolar range) interaction with nBgtS, which are expressed as high affinity radioiodinated Bgt binding sites by cells of the SH-SY5Y or IMR-32 human neuroblastomas. TPO and Bgt also share the capacity for high affinity interaction with muscle-type nAcChoR, which are expressed as high affinity binding sites for radioiodinated Bgt or tritium-labeled acetylcholine by cells of the TE671/RD human clone or the BC3H-1 mouse muscle cell line or on membrane preparations from Torpedo electroplax. TPO and Bgt act acutely as high affinity antagonists of muscle-type nAcChoR functional responses, which are measured using an isotopic rubidium ion efflux assay, on TE671/RD or BC3H-1 cells. In contrast, neither TPO nor Bgt are effective, at doses of up to 1 microM, as antagonists of ganglia-type nAcChoR function on SH-SY5Y or IMR-32 cells, nor are they potent as inhibitors of high affinity tritium-labeled acetylcholine binding to sites on putative ganglia-type nAcChoR expressed by SH-SY5Y or IMR-32 cells. These data indicate that some members of the extended nAcChoR family, including nBgtS and functional muscle-type nAcChoR but not ganglia-type nAcChoR, can interact with either Bgt or TPO. The results suggest that TPO may be an endogenous ligand active in both the nervous and immune systems and that some of its actions may be mediated via nBgtS or via functional blockade of muscle-type nAcChoR.

[The lymphocyte transformation test using mononuclear cells of elderly humans as a suitable in vitro method for testing of structure activity relationships of splenin peptides]. / Der Lymphozytentransformationstest unter Verwendung von mononukleären Zellen alter Menschen als geeignete in vitro-Methode zur Testung von Struktur-Wirkungs-Beziehungen von Splenin-Peptiden.

More and more new immunostimulatory substances are developed. Besides old and new drugs have to be examined with regard to their side effects. Hence it follows demands for effective test possibilities. The lymphocyte transformation test with mononuclear cells of old men is a suitable in vitro method for testing of substances with thymic hormone-like activity in order to investigate the structure-effect-relationships. Thymopentin, human splenopentin and human diacetyl-splenopentin increase the phytohaemagglutinin induced lymphocyte transformation, on the other hand in the case of bovine splenopentin and human splenotritin this effect was not observed. By means of a patient with hypogammaglobulinaemia is demonstrated that the lymphocyte transformation test could also be a suitable method for the estimation of pharmacodynamics such peptides after in vivo treatment.

Splenopentin (DAc-SP5)--influence on engraftment and graft-vs-host reaction after non-H-2 bone marrow transplantation in mice.

The influence of DAc-SP5 on engraftment and graft-vs-host reaction (GVHR) was studied in different non-H-2 strain combinations. The engraftment was more or less enhanced in every case, whereas the situation in the GVHR was completely different. In one case splenopentin did not influence the course of the GVHR so much, in a second case the symptoms of the GVHR were completely abolished, and in a third case the GVHR was dramatically enhanced up to a great mortality. Therefore, before application of DAc-SP5 to the bone marrow transplantation in humans in order to improve the engraftment, parameters have to be found which allow an exact prediction about the influence of splenopentin on the GVHR in each single case.

Thymopoietin inhibits function and ligand binding to nicotinic receptors at the neuromuscular junction.

Thymopoietin is a 48 to 49 amino acid polypeptide hormone of the thymus, which regulates immune function. The present experiments show that the polypeptide can cause a complete block of transmission of the phrenic nerve diaphragm junction of the rat in vitro; contractile responses evoked by phrenic nerve stimulation were blocked by concentrations of thymopoietin as low as 10(-8) M. The thymopoietin-induced inhibition of indirectly evoked muscle contractions was dose- and time-dependent, with the polypeptide being only slightly less potent than alpha-bungarotoxin (alpha-BGT). Twitch responses to direct electrical stimulation of the muscle were not affected by thymopoietin, indicating that it did not inhibit muscle tension by an action on the muscle contractile mechanism per se. Furthermore, thymopoietin did not alter resting or stimulated release of acetylcholine from the phrenic nerve, suggesting that it did not interact at a presynaptic level. On the other hand, thymopoietin inhibited the binding of [125I]alpha-BGT to the nicotinic receptor of rat hemidiaphragm. In intact muscle tissue, the IC50 value for inhibition of [125I]alpha-BGT binding by thymopoietin was 2.1 x 10(-7) M, a value similar to the concentration of polypeptide required to inhibit phrenic nerve-induced muscle contraction (IC50 value, 0.75-1.6 x 10(-7) M). In a muscle membrane preparation, the potency of thymopoietin to affect [125I]alpha-BGT binding was increased (IC50 value, 0.35 nM); thus thymopoietin has the potential to interact at the nicotinic receptor in the nM range. To conclude, the present results show that thymopoietin inhibits neuromuscular activity by an effect that appears to be a specific interaction at the nicotinic receptor.(ABSTRACT TRUNCATED AT 250 WORDS)

[Effect of humoral factors of the spleen after irradiation]. / Osobennosti deistviia gumoral'nykh faktorov selezenki pri obluchenii.

It is stated that therapeutic effect of humoral factors isolated from the cattle spleen is associated with its influence on alpha 2-macroglobulin (alpha 2-MG) performing a protective function. The results obtained permit recommending the isolated preparations to increase total resistivity of the organism under irradiation.