A. A. Zozulya

Identification of delta- and mu-type opioid receptors on human and murine dendritic cells.

The purpose of this study was to evaluate mu- and delta-opioid receptors (OR) on human and murine dendritic cells (DC). Expression of mu- and delta-OR mRNA on DC was demonstrated by RT-PCR. The immunocytochemical and Western blot analyses revealed the expression of OR protein in DC. Radioreceptor assay demonstrated the specific saturated temperature-dependent binding of [3H]-labeled opioid ligand on DC and B(max)=2.8+/-0.3 fmol/10(6) cells and K(D)=4.8+/-1.0 nM were calculated by a Scatchard analysis. Finally, OR ligands DADLE and DAGO dose-dependently modulated the capacity of DC to induce T cell proliferation in an MLR assay. Importantly, expression of functional OR on DC was significantly increased upon TNF-alpha-induced DC maturation. Thus, these data suggest a new mechanism of opioid-dependent neuroendocrine immunomodulation.

Personality, Coping Style, and Constitutional Neuroimmunology

Risk of developing certain diseases correlates with human personality. Cardiologists have defined Type “A” personalities as coronary-prone. Associated psychological peculiarities are easily angered, competitive, impatient and hard-driving. Psychologically-opposite people who are prone to suppress emotions and avoid conflicts (Type “C”), have a high risk of infectious diseases and certain forms of cancer. The development of contemporary biology and medicine determined an important role of the neuroendocrine and immune systems in these correlations. The peculiarity of human personality, as much as of animal behavioral patterns, is strongly expressed under stress conditions. The strategies of stress coping display a normal distribution in the human and wild animal populations, with truly passive and active coping styles located at the outermost regions of the curve. However, there are a number of strategies to breed experimental animals with extreme coping styles; animals selected for a passive coping style to acute stress show marked activation of the hypothalamic-pituitary-adrenal (HPA) axis and low stimulation of the sympathetic-adrenal system; both are associated with immunosuppression. An opposite reaction of the neuroendocrine system has been shown in animals with an active coping style to stress; this was associated with the signs of immunostimulation. Similarly, people with passive coping style (type “C”) might be at higher risk of infectious diseases and cancer, while people with active coping style (type “A”) might be predisposed to coronary, allergic, and autoimmune diseases. Furthermore, pain, decreased productivity, and anxiety, all common in patients with different diseases, are additional stressful entities. Thus, an adequate coping with a disease is an important approach to improve life quality and disease prognosis. Therefore, psychological and psychopharmacotherapeutic interventions that enhance effective coping should have beneficial effects in patients with immune-mediated diseases.

Autistic children display elevated urine levels of bovine casomorphin-7 immunoreactivity.

Elevated concentrations of circulating casomorphins (CM), the exogenous opioid peptides from milk casein, may contribute to the pathogenesis of autism in children. Because several mass spectrometry studies failed to detect casomorphins in autistic children, it was questioned whether these peptides can be detected in body fluids by mass spec. Here we demonstrated, using a novel high sensitivity ELISA method, that autistic children have significantly higher levels of urine CM-7 than control children. The severity of autistic symptoms correlated with concentrations of CM-7 in the urine. Because CMs interact with opioid and serotonin receptors, the known modulators of synaptogenesis, we suggest that chronic exposure to elevated levels of bovine CMs may impair early child development, setting the stage for autistic disorders.

Murine Dendritic Cells Express Functional Delta-Type Opioid Receptors

Dendritic cells (DC) are the most potent antigen-presenting cells (APC), and the only APC capable of presenting novel antigens to naïve T-cells. 1 Following their departure from the bone marrow, DC migrate to nonlymphoid tissues where they reside in an immature stage. The classical example is the epidermal Langerhans cell (LC) that recognizes and processes antigens in the skin. After migration to the secondary lymphoid organs, DC mature into cells that are capable of presenting antigens and stimulating T-cell proliferation. TNFα promotes DC maturation in vitro by upregulation of adhesion and costimulatory molecules, and downregulation of antigencapturing and antigen-processing molecules. DC are involved in the pathogenesis of numerous skin diseases including psoriasis, allergic contact eczema, atopic eczema, oral hairy leukoplakia, mycosis fungoides, and melanoma. The function of epidermal LC and dermal DC is likely to be regulated by neuropeptides released from nerves ending in the skin. In fact, receptors for calcitonin gene-related peptide, 2 pituitary adenylate cyclase activating polypeptide, 3 gastrin-releasing peptide, 3 and substance P 4 have been identified on epidermal LC. The aim of this work was to evaluate presence and function of δ -type opioid receptors (DOR) on DC.

Reactions between beta-casomorphins-7 and 5-HT2-serotonin receptors.

Radioreceptor analysis showed that human β-casomorphin-7 displaced 3H-spiperone from 5-HT2-serotonin receptors of the rat cerebral frontal cortex: EC50 8±1 μM. Human and bovine β-casomorphin-7 dose-dependently blocked serotonin-induced human platelet aggregation: IC50 5±1 and 20±4 μM, respectively. It was proved that β-casomorphins-7 act as 5-HT2-serotonin receptor antagonists; one of the mechanisms of their biological effects is presumably associated with modulation of the serotoninergic system.

[Semax and selank inhibit the enkephalin-degrading enzymes from human serum]].

Dose-dependent effect of synthetic heptapeptides Semax (Met-Glu-His-Phe-Pro-Gly-Pro) and Selank (Thr-Lys-Pro-Arg-Pro-Gly-Pro) on the enkephalin-degrading enzymes of human serum was demonstrated. The inhibitory effects of Semax (IC5010 μM) and Selank (IC5020 μM) are more pronounced than that of puromycin (IC5010 mM), bacitracin, and some other inhibitors of peptidases. Beside the heptapeptides, their pentapeptide fragments also possessed an inhibitory effect; tri-, tetra- and hexapeptide fragments did not display such an effect. As the above enzymes take part in degradation of not only enkephalins but also other regulatory peptides, it can be assumed that one of the mechanisms of biological activity of Semax and Selank is related to this inhibitory activity of theirs.

Evenly tritium labeled peptides in study of peptide in vivo and in vitro biodegradation

Biologically active peptides evenly labeled with tritium were used for studying the in vitro and in vivo biodegradation of the peptides. Tritium-labeled peptides with a specific radioactivity of 50–150 Ci/mmol were obtained by high temperature solid phase catalytic isotope exchange (HSCIE) with spillover tritium. The distribution of the isotope label among all amino acid residues of these peptides allows the simultaneous determination of practically all possible products of their enzymatic hydrolysis. The developed analytical method includes extraction of tritium-labeled peptides from organism tissues and chromatographic isolation of individual labeled peptides from the mixture of degradation products. The concentrations of a peptide under study and the products of its biodegradation were calculated from the results of liquid scintillation counting. This approach was used for studying the pathways of biodegradation of the heptapeptide TKPRPGP (Selank) and the tripeptide PGP in blood plasma. The pharmacokinetics of Selank, an anxiolytic peptide, was also studied in brain tissues using the intranasal in vivo administration of this peptide. The concentrations of labeled peptides were determined, and the pentapeptide TKPRP, tripeptide TKP, and dipeptides RP and GP were shown to be the major products of Selank biodegradation. The study of the biodegradation of the heptapeptide MEHFPGP (Semax) in the presence of nerve cells showed that the major products of its biodegradation are the pentapeptide HFPGP and tripeptide PGP. The enkephalinase activity of blood plasma was studied with the use of evenly tritium labeled [Leu]enkephalin. A high inhibitory effect of Semax on blood plasma enkephalinases was shown to arise from its action on aminopeptidases. The method, based on the use of evenly tritium-labeled peptides, allows the determination of peptide concentrations and the activity of enzymes involved in their degradation on a μg scale of biological samples both in vitro and in vivo.

The Inhibitory Effect of Selank on Enkephalin-Degrading Enzymes as a Possible Mechanism of Its Anxiolytic Activity

Examination of patients with various forms of anxiety and phobic disorders (according to DSM-4 criteria) demonstrated a considerable shortening of enkephalin half-life and reduced total enkephalinase activity in the blood during generalized anxiety, but not during panic disorders and agoraphobia. This was probably related to low blood concentration of endogenous inhibitors of enkephalin-degrading enzymes in patients with generalized anxiety disorders. Heptapeptide Selank (Thr-Lys-Pro-Arg-Pro-Gly-Pro), which attenuates behavioral anxiety reactions and does not cause side effects typical of most anxiolytics, dose-dependently inhibited enzymatic hydrolysis of plasma enkephalin (IC50 15 μM). Selank was more potent than peptidase inhibitors bacitracin and puromycin in inhibiting enkephalinases. These results suggest that high efficiency of Selank in the therapy of anxiety and phobic disorders, including generalized anxiety, is due to its ability to inhibit enkephalin hydrolysis.

Leu-Enkephalin Generally Labeled with Tritium in Studying the Selank Inhibiting Effect on the Enkephalin-Degrading Enzymes of Human Blood Plasma

A method of analysis of enkephalinase activity in blood plasma based on the application of Leu-enkephalin generally labeled with tritium at all its amino acid residues was developed. The method allows the simultaneous estimation of activity of several peptidases in microquantities of tissues. [G-3H]Leu-enkephalin was prepared by the method of solid phase catalytic isotope exchange (120 Ci/mmol) and subjected to proteolysis by the treatment with blood plasma. The resulting radioactive metabolites were separated by HPLC in the presence of the mixture of unlabeled fragments of Leu-enkephalin as internal standards. It was shown that aminopeptidases, dipeptidylaminopeptidases, and dipeptidylcarboxypeptidases respond for approximately 80%, 2%, and 10% of the total enzymatic activity, respectively. The new pathway of degradation of Leu-enkephalin by carboxypeptidase that provides for ∼6% of the total enkephalin-degrading activity was discovered. Bestatin was shown to predominantly inhibit aminopeptidases and carboxypeptidases, whereas selank is more specific for carboxypeptidases and dicarboxypeptidases.

Influence of human B-casomorphin-7 on specific binding of 3H-spiperone to the 5-HT2-receptors of rat brain frontal cortex.

Using radio-receptor analysis, it has been demonstrated that human beta-casomorphin-7 (Tyr-Pro-Phe-Val-Glu-Pro-Ile) displaces 3H-spiperone from 5-HT2-receptors of rat brain frontal cortex. IC50 of human beta-casomorphin-7 was 8 microM. These data suggest that one of the mechanisms of neurotropic action of beta-casomorphin-7 is might be associated with its influence on the serotoninergic system.