Effects of short peptides on thymocyte blast transformation and signal transduction along the sphingomyelin pathway

Immunomodulating effects of synthetic peptides Vilon (Lys-Glu), Epithalon (Ala-Glu-Asp-Gly), and Cortagen (Ala-Glu-Asp-Pro) and possible involvement of the sphingomyelin signal transduction pathway in their effects in mouse thymocytes were studied. Vilon produced the most potent comitogenic effect on thymocyte proliferation and modulated comitogenic activity of interleukin-1b. Epithalon was less potent, while Cortagen produced no such effects. Vilon produced a more pronounced stimulatory effect on sphingomyelinase activity in mouse thymocyte membranes compared to Epithalon and Cortagen.     

Tissue-Specific Effects of Peptides

Synthetic peptides (cytogens) Cortagen, Epithalon, Livagen, and Vilon stimulated the growth of explants from rat brain cortex, subcortical structures, liver, and thymus, respectively, in organotypic cultures. These peptides produced tissue-specific effects: they stimulated the growth of explants from tissues, whose cytomedins (peptide complexes) were used for chemical synthesis.     

Effects of Short Peptides on Lymphocyte Chromatin in Senile Subjects

Effects of synthetic short peptides (Vilon, Epithalon, Livagen, Prostamax, and Cortagen) on activity of ribosome genes, parameters of common heterochromatin melting, polymorphism of structural heterochromatin (C segments) of chromosomes 1, 9, and 16, and variability of facultative heterochromatin were studied in leukocytes of subjects aged 75-88 years. All the studied peptides induced activation of ribosome genes, decondensation of densely packed chromatin fibrils, and release of genes repressed as a result of age-specific condensation of the cellular euchromatin regions (deheterochromatinization of facultative chromatin). Treatment with Epithalon, Livagen, and Prostamax led to decondensation of chromosome 1 pericentromeric structural chromatin, while Epithalon and Livagen treatment led to changes in chromosome 9 as well. Hence, short peptides activate heterochromatin and heterochromatinized regions of cell chromosomes in senile subjects.     

Synthesis of IL-2 mRNA in Cells of Rat Hypothalamic Structures after Injection of Short Peptides

In situ hybridization on paraffin sections of the rat brain showed that synthetic peptides Vilon, Epithalon, and Cortagen modulated the expression of IL-2 gene in vivo in cells of some hypothalamic structures depending on the terms and routes of administration.__________Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 139, No. 6, pp. 688–690, June, 2005     

Production of lymphocyte-activating factors by mouse macrophages during aging and under the effect of short peptides

Age-specific characteristics of production of lymphocyte-activating factor by mouse peritoneal macrophages and modulation of this production by short synthetic peptides (Vilon, Epithalon, and Cortagen) were studied. The production of lymphocyte-activating factors by macrophages stimulated with lipopolysaccharides in vitro was lower in old animals. The opposite modulating effects of short peptides on the production of lymphocyte-activating factors by resident and lipopolysaccharide-stimulated macrophages in young and old mice were demonstrated for the first time. This is a possible mechanism of immune system dysfunction during aging, which opens new vistas for its correction with short synthetic peptides. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 142, No. 9, pp. 333–336, September, 2006     

Studies of the effects of Vilon and Epithalon on gene expression in mouse heart using DNA-microarray technology

Expression of 15,247 clones from a cDNA library in the heart of mice receiving Vilon and Epithalon was studied by DNA-microarray technology. We revealed 300 clones (1.94% of the total count), whose expression changed more than by 2 times. Vilon changed expression of 36 clones, while Epithalon modulated expression of 98 clones. Combined treatment with Vilon and Epithalon changed expression of 144 clones. Vilon alone or in combination with Epithalon activated expression of 157 clones (maximally by 6.13 times) and inhibited expression of 23 clones (maximally by 2.79 times). Epithalon alone or in combination with Vilon activated expression of 194 clones (maximally by 6.61 times) and inhibited expression of 48 clones (maximally by 2.71 times). Our results demonstrate the specific effects of Epithalon and Vilon on gene expression.     

Effect of Vilon and Epithalon on glucose and glycine absorption in various regions of small intestine in aged rats

Vilon (Lys-Glu) and Epithalon (Ala-Glu-Asp-Gly) administered orally for 1 month improved transport characteristics of the small intestine in aged rats. Vilon enhanced passive glucose accumulation in the serous fluid in inverted sac made from the distal region of the small intestine, while Epithalon enhanced this process in the medial region. Vilon stimulated active glucose accumulation in the serous sac of the medial small intestine, Epithalon - in the proximal and distal small intestinal segments. Glycine absorption increased only in the proximal intestinal segment under the effect of Epithalon.     

Interleukin-2 concentration in hypothalamic structures of rats receiving peptides during mild stress

The number of hypothalamic IL-2-containing cells changed in rats receiving Vilon and Epithalon during mild stress (handling). The number of IL-2-positive cells in hypothalamic structures decreased 24 h after intramuscular injection of Epithalon and 2 h after intranasal administration of the test peptides. Adaptation of animals to experimental conditions prevented the decrease in the number of IL-2-positive cells in the supraoptic nucleus after intranasal administration of Epithalon. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 141, No. 4, pp. 371–374, April, 2006     

Epithalon inhibits tumor growth and expression of HER-2/neu oncogene in breast tumors in transgenic mice characterized by accelerated aging

Female transgenic FVB mice carrying breast cancer gene HER-2/neu were monthly injected with Vilon or Epithalon (1 g subcutaneously for 5 consecutive days) starting from the 2nd month of life. Epithalon markedly inhibited neoplasm development: the maximum size of breast adenocarcinomas was 33% lower than in the control (p<0.05). The intensity of HER-2/neu mRNA expression in breast tumors of Epithalon-treated mice was 3.7 times lower than in control animals. These results indicate that Epithalon inhibits breast tumor development in transgenic mice, which is probably related to suppression of HER-2/neu expression.     

Expression of Argyrophilic Proteins in the Nucleolar Organizer Regions of Human Thymocytes and Thymic Epitheliocytes under Conditions of Coculturing with Vilon and Epithalon Peptides

Vilon stimulated and Epithalon suppressed the expression of argyrophilic proteins in nucleolar organizer regions of thymocytes and epithelial cells, stimulating or reducing, respectively, the formation, assembly, and transport of ribosomes into the cytoplasm and thus determining the intensity of protein synthesis in these cells. A direct mitogenic effect of Vilon was also revealed: this peptide promoted thymocyte transformation into proliferating blast cells.     

Effects of Peptides on Generation of Reactive Oxygen Species in Subcellular Fractions of Drosophila Melanogaster

We studied the effects of Epithalon (Ala-Glu-Asp-Gly) and Vilon (Lys-Glu) on free radical processes in highly inbred HA⁺line of Drosophila melanogaster. Vilon inhibited generation of reactive oxygen species in mitochondria, but stimulated this process in the cytosol. We found sex- and age-related differences in the generation of reactive oxygen species and cytosol antioxidant activity.     

Epithalon decelerates aging and suppresses development of breast adenocarcinomas in transgenic her-2/neu mice

Female transgenic FVB/N mice carrying the breast cancer gene HER-2/neu received epithalon (Ala-Glu-Asp-Gly) in a dose of 1 mg subcutaneously 5 times a week to from the 2nd month of life to death. Epithalon prolonged the average and maximum lifetimes of mice by 13.5 (p<0.05) and 13.9%, respectively. The peptide prolonged the average lifetime of animals without neoplasms (by 34.2%, p<0.05). Epithalon decelerated the development of age-related disturbances in reproductive activity and suppressed the formation of neoplasms. The peptide decreased the incidence of breast adenocarcinomas, lungs metastases (by 1.6 times, p<0.05), and multiple tumors (by 2 times). Epithalon 3.7-fold increased the number of mice without breast tumors (p<0.05), while the number of animals with 6 or more breast tumors decreased by 3 times (p<0.05). Epithalon prolonged the lifetime of mice with breast tumors by 1.4 times (p<0.05). These results indicate that Epithalon possesses geroprotective activity and inhibits breast carcinogenesis in transgenic mice, which is probably related to suppression of HER-2/neu expression.     

Two opposing modes of action of beta-endorphin on lymphocyte function

Five opioid peptides (alpha-, beta-, and gamma-endorphin, methionine- and leucine-enkephalin) were tested for their effect on the concanavalin A-induced proliferative response of splenocytes of adult male F344 rats. The continuous presence of these opioid peptides during culture of T cells did not affect proliferation. However, 30 min of preincubation with beta-endorphin (beta-end), but not with the other opioid peptides, resulted in a dose-dependent enhancement of proliferation of 50-100%. This potentiating effect of beta-end on proliferation was preceded by an increase in the production of interleukin-2 (IL-2) and in the extent of IL-2 receptor expression. The stimulatory effect of beta-end was not prevented by naloxone, indicating that classical opioid receptors were not involved. The continuous presence of beta-end (or alpha-end) in cultures of cells that had been preincubated with beta-end completely abolished the stimulatory effect, pointing towards the potential of beta-end to regulate T-cell function via different mechanisms.     

Effect of Transplantation of Splenic Lymphoid Cells on Functional Activity of the Immune and Nervous System in Experimental Animals

We studied the effect of transplantation of splenic lymphoid cells on functional activity of the nervous (orientation and exploratory behavior and expression of genes for interleukin-1β, type 1 interleukin-1 receptor, and erythropoietin in the brain) and immune system (cellular and humoral immune response, proliferative activity of immunocompetent cells, and expression of cytokine genes in splenocytes) in syngeneic animals with different behavioral characteristics. Intravenous injection of the lymphoid fraction of splenocytes from donor mice with a certain behavioral pattern in the open-field test had a modulatory effect on vertical locomotor activity of syngeneic recipient mice. The increase or decrease in vertical locomotor activity due to transplantation of immunocompetent cells was accompanied by specific changes in mRNA level for erythropoietin receptor and type 1 interleukin-1 receptor in brain cells of recipient mice. The regulation of orientation and exploratory behavior was also accompanied by changes in functional activity of the immune system in recipient animals. It was manifested in modulation of proliferative activity of thymic and splenic cells and cytokine gene expression in splenocytes.     

Role of the transgenic human thyrotropin receptor A-subunit in thyroiditis induced by A-subunit immunization and regulatory T cell depletion

Transgenic BALB/c mice that express intrathyroidal human thyroid stimulating hormone receptor (TSHR) A-subunit, unlike wild-type (WT) littermates, develop thyroid lymphocytic infiltration and spreading to other thyroid autoantigens after T regulatory cell (T(reg)) depletion and immunization with human thyrotropin receptor (hTSHR) adenovirus. To determine if this process involves intramolecular epitope spreading, we studied antibody and T cell recognition of TSHR ectodomain peptides (A-Z). In transgenic and WT mice, regardless of T(reg) depletion, TSHR antibodies bound predominantly to N-terminal peptide A and much less to a few downstream peptides. After T(reg) depletion, splenocytes from WT mice responded to peptides C, D and J (all in the A-subunit), but transgenic splenocytes recognized only peptide D. Because CD4(+) T cells are critical for thyroid lymphocytic infiltration, amino acid sequences of these peptides were examined for in silico binding to BALB/c major histocompatibility complex class II (IA-d). High affinity subsequences (inhibitory concentration of 50% < 50 nm) are present in peptides C and D (not J) of the hTSHR and mouse TSHR equivalents. These data probably explain why transgenic splenocytes do not recognize peptide J. Mouse TSHR mRNA levels are comparable in transgenic and WT thyroids, but only transgenics have human A-subunit mRNA. Transgenic mice can present mouse TSHR and human A-subunit-derived peptides. However, WT mice can present only mouse TSHR, and two to four amino acid species differences may preclude recognition by CD4+ T cells activated by hTSHR-adenovirus. Overall, thyroid lymphocytic infiltration in the transgenic mice is unrelated to epitopic spreading but involves human A-subunit peptides for recognition by T cells activated using the hTSHR.     

Comparative analysis of the expression of c-Fos and interleukin-2 proteins in hypothalamus cells during various treatments

The aim of the present work was to perform a combined analysis of the degree of activation of the anterior hypothalamus of the rat and expression of the interleukin-2 gene during treatments of different types: mild stress (“handling”) and adaption to it, as well as intranasal administration of physiological saline and the peptides Vilon (Lys-Glu) and Epitalon (Ala-Glu-Asp-Gly). Changes in the numbers of c-Fos-and IL-2-positive cells in structures of the lateral area (LHA) and anterior (AHN), supraoptic (SON), and paraventricular (PVN) nuclei of the hypothalamus in Wistar rats. Ratios of the quantities of c-Fos-and IL-2-positive cells were determined in intact animals and after activation of brain cells initiated by different treatments; the influences of adaptation to handling on the nature of changes in the expression of these proteins was also studied. Combined analysis of the intensity of expression of these two proteins — c-Fos, a marker of neuron activation and a trans-factor for the IL-2 cytokine gene and other inducible genes, and IL-2 — in intact animals and after various treatments showed that the process of cell activation in most of the hypothalamic structures studied correlated with decreases in the quantity of IL-2-positive cells in these structures; different patterns of changes in the numbers of c-Fos-and IL-2-positive cells were seen in response to different treatments in conditions of stress and adaptation to it. __________ Translated from Rossiiskii Fiziologicheskii Zhurnal imeni I. M. Sechenova, Vol. 93, No. 2, pp. 150–160, February, 2007.     

2-Arachidonoyl-glycerol suppresses interferon-gamma production in phorbol ester/ionomycin-activated mouse splenocytes independent of CB1 or CB2

2-Arachidonoyl-glycerol (2-AG), an endogenous ligand for cannabinoid receptor types 1 and 2 (CB1 and CB2), has previously been demonstrated to modulate immune functions including suppression of interleukin-2 expression and nuclear factor of activated T cells (NFAT) activity. The objective of the present studies was to investigate the effect of 2-AG on interferon-gamma (IFN-gamma) expression and associated upstream signaling events. Pretreatment of splenocytes with 2-AG markedly suppressed phorbol 12-myristate 13-acetate plus calcium ionophore (PMA/Io)-induced IFN-gamma secretion. In addition, 2-AG suppressed IFN-gamma steady-state mRNA expression in a concentration-dependent manner. To unequivocally determine the putative involvement of CB1 and CB2, splenocytes derived from CB1(-/-)/CB2(-/-) knockout mice were used. No difference in the magnitude of IFN-gamma suppression by 2-AG in wild-type versus CB1/CB2 null mice was observed. Time-of-addition studies revealed that 2-AG treatment up to 12 h post-cellular activation resulted in suppression of IFN-gamma, which was consistent with a time course conducted with cyclosporin A, an inhibitor of NFAT activity. Coincidentally, 2-AG perturbed the nuclear translocation of NFAT protein and blocked thapsigargin-induced elevation in intracellular calcium, suggesting that altered calcium regulation might partly explain the suppression of NFAT nuclear translocation and subsequent IFN-gamma production. Indeed, Io partially attenuated the 2-AG-induced suppression of PMA/Io-stimulated IFN-gamma production. Taken together, these data demonstrate that 2-AG suppresses IFN-gamma expression in murine splenocytes in a CB receptor-independent manner and that the mechanism partially involves suppression of intracellular calcium signaling and perturbation of NFAT nuclear translocation.     

Synthetic peptides corresponding to the F protein of RSV stimulate murine B and T cells but fail to confer protection

Summary We have previously located a major neutralization site of the fusion protein of respiratory syncytial virus (RSV) in the polypeptide region extending from amino acids Ile221 to Glu232. In this report, 8 peptides corresponding to the six major hydrophilic regions of the F₁ subunit were selected to analyse their immunogenic and protective capacities as well as their ability to block the high neutralization activities of 4 monoclonal antibodies (MAbs). Only 5 of the 8 peptides tested induced specific antibodies while all induced an in vitro interleukin-2 response of splenocytes from immunized mice. Peptide 3 (Ile221-Phe237) was able to elicit neutralizing antibodies, confirming our previous hypothesis concerning the location of a neutralization site. However, immunization with the latter did not induce significant reduction of virus in lungs of BALB/c mice upon challenge, probably due to an inadequate level of circulating neutralizing antibodies. Interestingly, peptides 2 (Asn216-Glu232), 3 (Ile221-Phe237), and 5 (Ser275-Ile288) blocked in vitro neutralization by four different F₁ specific MAbs. A hypothesis is proposed to explain these results.     

Biological activity of analogs of the peptide hormone luliberin in the regulation of the immune response of T cells

The ability of the antitumor analogs of luliberin (LH-RH), a hypothalamic peptide hormone, to stimulate the immune function of T cells is examined in experiments with the gene coding for interleukin-2 (IL-2). Recombinant MIL2C or 4×Pu DNA containing the marker gene of chloramphenicol acetyltransferase (CAT) under the control of a 2.2 kb promoter of murine IL-2 gene or four copies of purine-rich element (from −292 to −246 base pairs), respectively, is injected inXenopus laevis oocytes. The promoter activity is blocked by inoculation of the protein fraction of nuclear extracts from resting mouse splenic T cells. The IL-2 gene promoter is dereppressed after injection of the short LH-RH analog L1 (7 amino acid residues) into the oocyte nucleus or cytoplasm. The addition of L1 or L2 (an LH-RH analog consisting of 10 amino acid residues) to the incubation medium activates mouse splenic T cells and stimulates the synthesis of IL-2 mRNA 2- to 3-fold more intensely than ConA+rIL-2, judging from dot-blot andin situ hybridization data. Cytological analysis of cell culture shows that the presence of L1 and L2 peptides in the culture medium promotes differentiation of T cells. It is hypothesized that the antitumor activity of these peptides is associated with the stimulation of IL-2 synthesis. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 122, No. 9, pp. 334–337, September, 1996