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.

Use of tilapia piscidin 3 (TP3) to protect against MRSA infection in mice with skin injuries.

Antimicrobial peptides (AMPs), represent promising agents for new therapeutic approaches of infected wound treatment, on account of their antimicrobial and wound closure activities, and low potential for inducing resistance. However, therapeutic applications of these AMPs are limited by their toxicity and low stability in vivo. Previously, we reported that the 23 amino-acid designer peptide TP3 possessed antimicrobial activities. Here, we analyzed the wound closure activities of TP3 both and in vivo. TP3 at doses of up to 40 µg/ml did not affect the viability of baby hamster kidney cells. Furthermore, TP3 was found to be highly effective at combating peritonitis and wound infection caused by MRSA in mouse models, without inducing adverse behavioral effects or liver or kidney toxicity. TP3 treatment increased survival by 100% at 8 days after infection, and accelerated the progression of proliferation, remodeling, and maturation of infected wounds. Taken together, our results indicate that TP3 enhances the rate of survival of mice infected with the bacterial pathogen MRSA through both antimicrobial and immunomodulatory effects. Overall, these results suggest that TP3 may be suitable for development as a novel topical agent for treatment of infected wounds.

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.

Synthesis of [I-NaI3]] thymopoietin II and examination of its immunological effect on the uremic T-lymphocytes.

A TP II analogue, [1-Nal3] TP II, was synthesized by a conventional solution method, followed by deprotection with 1M TFMSA-thioanisole (molar ratio 1:1) in TFA in the presence of Me2Se and m-cresol as scavengers. The synthetic [1-Nal3] TP II, TP II and [Phe (4 F)3] TP II were tested for comparative effect on the impaired T-lymphocyte transformation by PHA in uremic patients suffering from recurrent infectious diseases. The synthetic analogue was found to have stronger restorative activity than those of our synthetic TP II and [Phe (4F)3] TP II.

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.

Stimulation of IL-2 production and CD2R expression by splenopentin analogs.

Splenopentin (SP-5), is a pentapeptide corresponding to the amino acid sequence 32-36 (Arg-Lys-Glu-Val-Tyr) of the splenic hormone splenin. Its synthetic analogs: Lys-Lys-Glu-Val-Tyr(1) and D-Lys-Lys-Glu-Val-Tyr (2) have been evaluated for active T-cell rosette (CD2R), total T-cell rosette (CD2), interleukin-2 (IL-2) stimulation and effect on antibody production. SP-5 as well as both the analogs stimulated CD2R. Analogs (1) and (2) were also found to stimulate IL-2 production. These observations suggest that in vitro human NK cell augmentation with analogs (1) and (2) reported earlier may be due to enhanced IL-2 production.

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.

[Role of the thymus and normal microbial flora on plasma fibronectin concentration in mice treated with immunomodulatory drugs]. / A thymus és a mikrobaflóra plazma fibronektin szintet befolyásoló szerepének vizsgálata immunmoduláns készítményekkel kezelt egerekben.

The plasma fibronectin (pFN) concentration (cc)-determined by electro-immunodiffusion method-of untreated genetically or artificially athymic mice, or treated with TP-4 (thymus hormone sequence analog synthetic preparation) showed no significant difference from their euthymic or untreated controls. In contrast, the pFN cc in mice with different microbiological state showed significant alterations; the highest level occurred in conventional mice. The lower level in germfree mice was increased by bacterial monocontamination. The alternation from SPF into conventional state in athymic mice or treatment of athymic and euthymic mice with Bordetella pertussis vaccine also resulted in the increase of the pFN cc. Based on these and earlier results, it was assumed that the pFN cc is independent from the presence or absence of the thymus, but it depends on the actual microbiological state of the macroorganism.

Synthesis of [Phe(4F)3]thymopoietin II and examination of its immunological effect on the impaired blastogenic response of T-lymphocytes of uremic patients.

[Phe(4F)3]thymopoietin II was synthesized using a conventional solution method. The deprotection of the protected [Phe(4F)3]thymopoetin II was achieved by treatment with 1 M trifluoromethanesulfonic acid:thioanisole (molar ratio 1:1) in trifluoroacetic acid in the presence of dimethylselenide and m-cresol. The synthetic f1p4(4F)3]thymopoietin II and thymopoietin II were tested for effect on impaired T-lymphocyte transformation by phytohemagglutinin in uremic patients suffering from recurrent infectious diseases. The restoring activity on the impaired phytohemagglutinin stimulation of T-lymphocytes was obtained after incubation of peripheral lymphocytes isolated from uremic patients with the synthetic [Phe(4F)3]thymopoietin II. This peptide exhibited far stronger restoring effect than that of our synthetic thymopoietin II.

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.

Detection of thymopoietin-responsive proteins in nude mouse spleen cells by two-dimensional polyacrylamide gel electrophoresis and image processing.

Lymphoid cells isolated from the spleen of BALB/c nu/nu nude mice were treated with synthetic human thymopoietin, and newly synthesized proteins were labeled by [35S]methionine incorporation. In the control experiment, the same lot of spleen cells were incubated in the labeling medium without the addition of thymopoietin. Urea/detergent-soluble proteins were extracted from the cells after 3 h incubation to be separated by two-dimensional poly-acrylamide gel electrophoresis. Spots of [35S]methionine-labeled proteins were visualized by autoradiography and analyzed by image processing. The computer-aided spot matching screened out three major thymopoietin-responsive proteins, TRP-1, -2 and -3. [35S]Methionine incorporation into TRP-3, of which the isoelectric point and molecular mass were approximately pI 5 and 10 kDa, respectively, was decreased by the thymopoietin treatment. In contrast with the down regulation, TRP-1, which was slightly higher in pI and slightly larger in molecular mass, and TRP-2, which was slightly higher in pI and almost the same in molecular mass as TRP-3, were evidently induced by the treatment. However, TRPs could not be assigned to Thy-1 antigen on the difference in molecular mass. The specific induction by the thymopoietin treatment suggested that TRP-1 and -2 might be novel proteins related to the intracellular signal transduction.

[Natural factor splenin and mechanisms of its action]. / Pryroda aktyvnoho chynnyka spleninu ta mekhanizm ioho dii.

The search of biologically active chemical compounds of the drug was carried out basing on the revealed lymphocytotropic property of splenin. A non-protein factor of the nucleoside origin was isolated from splenin by precipitation with mercury. It had a pronounced direct mitogenic effect on splein and thymic T-lymphocetes, being an active component of the drug. This factor increases the mass of lymphoid organs, promotes restoration of postradiation and steroidogenic lymphocytopenias.

[Role of non-protein factor of splenin in the regulation of antibody formation]. / Rol nebilkovoho chynnyka spleninu v rehuliatsii antytilohenezu.

Non-protein factor of splenin that possesses T-mitogen activity under experimental conditions reproduces immunomodulatory action of the native drug in what concerns regulation of immunoglobulin synthesis. Premedication of CBA mice with the isolated factor increases primary immune response to ram erythrocytes by 64%. When immediate allergic reaction develops in Wistar rats and BALB/c mice this factor suppresses IgE-antibody production via stimulation of suppressors T-cells.

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.

The effects of synthetic thymopoietin on binding of alpha-bungarotoxin to rat neural membranes.

Chemically synthesized bovine thymopoietin I (TPO-I) and thymopoietin II (TPO-II) were evaluated as inhibitors of 125I alpha-Bungarotoxin binding to rat brain neural membranes and found to be over 1,000 x less potent (IC50 = 10 microM) than reported for thymopoietin isolated from bovine thymus.