Differentiation of human macrophages with anaphylatoxin C3a impairs alternative M2 polarization and decreases lipopolysaccharide-induced cytokine secretion.

Anaphylatoxin C3a is a small signaling polypeptide that is generated during complement activation. C3a is involved in the regulation of various innate and adaptive immune system processes; however, the role of C3a in macrophage differentiation and polarization is poorly elucidated. Here we showed that C3a impairs alternative M2 polarization of human macrophages and suppressed CD206, IL1Ra and CCL22 expression. C3a leads to a decrease of nuclear receptor PPARγ expression via the ERK1/2 signaling pathway, resulting in repressed PPARγ-dependent activation of CD36, FABP4 and LXRα genes and blunted response to an LXR ligand TO901317. Using small interfering RNA and agonist/antagonist approaches we showed that C3a decreases CD206, IL1Ra and CCL22 transcription at least partly in a PPARγ-dependent manner in M2 macrophages. Moreover, C3a impairs efferocytosis by M2 macrophages and inhibits their migratory activity. By contrast, macrophages treated with C3a during differentiation show blunted response to lipopolysaccharide stimulation owing to downregulation of TLR4 and lipid raft content. At the same time, differentiation of macrophages with C3a does not change M1 polarization in interferon gamma (IFNγ) and IFNγ + lipopolysaccharide-treated macrophages. These data provide a novel role of complement system and C3a in the regulation of M2 macrophage polarizations and suggest crosstalk between C3a, TLR4, PPARγ and LXR signaling pathways.

A novel method for purification of biologically active C-terminal fragment of human recombinant anti-mullerian hormone.

Anti-mullerian hormone (AMH) is one of the least studied members of transforming growth factor beta superfamily showing pro-apoptotic activity against cells positive for hormone type II receptor overexpressed by malignant cells in many cancer cases. Here, we propose an improved method for isolation of recombinant C-terminal AMH fragment (C-rAMH) to obtain homogeneous preparations of this protein with high biological activity. In contrast to our previously developed C-rAMH purification technology based on reversed-phase HPLC, the key stage of the new approach is hydrophobic interaction chromatography using Toyopearl Butyl-650S resin performed under more benign conditions. This modification of the previously developed method allowed highly purified C-rAMH to be obtained that is characterized by twice the specificity estimated as the ability to bind to the recombinant analog of AMH type II receptor and by significantly higher biological activity, that is, the ability to induce the death of target cells. Thus, we made the purification technology even more cost-effective and suitable for the production of drug forms based on C-rAMH.

Epitope specificity of two anti-morphine monoclonal antibodies: In vitro and in silico studies.

Monoclonal antibodies (mAbs) against morphine are important in the development of immunotherapeutic and diagnostic methods for the treatment and prevention of drug addiction. By the surface plasmon resonance (SPR) and enzyme immunoassay techniques, we characterized two previously obtained mAbs 3K11 and 6G1 and showed their ability to recognize free morphine and morphine-containing antigens in different ways because of the epitope specificity thereof. Using the defined amino acid sequences, we obtained three-dimensional models of the variable regions of Fab fragments of these antibodies and compared them with the known sequence and spatial structure of the anti-morphine antibody 9B1. Docking simulations are performed to obtain models of the antibodies complexes with morphine. Differences in the models of 3K11 and 6G1 complexes with morphine correlate with their experimentally detected epitope specificity. The results, in particular, can be used for the structure-based design of the corresponding humanized antibodies. According to our modeling and docking results, the very different modes of morphine binding to mAbs 3K11 and 6G1 are qualitatively similar to those previously reported for cocaine and two anti-cocaine antibodies. Thus, the obtained structural information brings more insight into the hapten recognition diversity.

Purification of human recombinant anti-mullerian hormone and its derivatives.

Anti-mullerian hormone (AMH) is a cytokine of transforming growth factor ß (TGF-ß) superfamily able to induce apoptosis in cells bearing specific AMH type II receptors (AMHRII). AMHRII is overexpressed in some malignant cells, so at present recombinant AMH (rAMH) is considered as a new candidate antineoplastic drug. The use of rAMH may be especially effective in case of such severe diseases as ovarian, prostate and breast cancer. However, the development of a new drug is hampered by the laboriousness of obtaining highly purified rAMH and by the lack of data about the pharmacological characteristics of rAMH derivatives. In this work, we obtained preparations of prohormone, half-cleaved rAMH and a C-terminal fragment of rAMH, which was confirmed by qualitative and quantitative analyses. To obtain rAMH and its derivatives we used a previously developed highly effective producer strain containing the optimized human AMH gene. The production process has been divided into several stages: (a) rAMH biosynthesis in the bioreactor; (b) culture media preparation; (c) purification of rAMH and its derivatives using immunoaffinity chromatography and reversed-phase HPLC; (d) identification of the purified proteins by immunoblotting and analytical reversed-phase HPLC; and (e) evaluation of the hormone forms activity. The obtained proteins may be used in preclinical trials and in vitro study of rAMH derivatives properties.

Hormonal function and proliferative activity of thymic cells in humans: immunocytochemical correlations.

OBJECTIVES: The immunocytochemical study of the localization of hormones in thymic cells has been performed to clarify possible correlations of their expression with proliferative activity of thymocytes. METHODS: We used commercial antibodies to serotonin, melatonin, somatostatin, glucagon, gastrin, beta-endorphin and histamine, and ABP or BSP kits for visualization of reaction. Computer image analysis was used to find correlations between hormone production and proliferative activity of thymocytes. RESULTS: Different subpopulations of thymocytes are able to produce hormones: precursors of T-lymphocytes (CD4-CD8-) contain serotonin and melatonin, immature cortical cells (CD4+CD8+) produce only serotonin, mature medullar cells (CD4+CD8-) show immunoreactivity to serotonin, melatonin, beta-endorphin and histamine. The expression of serotonin, somatostatin and gastrin is localized in thymic epithelial cells, formatting Gassal's bodies. Proliferative activity of thymocytes depends from the expression of serotonin and somatostatin in thymic cells. CONCLUSION: The data received testify the expression of different hormones in human thymic cells and showing by this fact high endocrine activity of thymus. The presence of correlations between hormonal expression and cell proliferative activity could be considered as the bright illustration of important role of neuroimmunoendocrine mechanisms in the regulation of local thymic homeostasis.

Gastrointestinal melatonin: cellular identification and biological role.

Melatonin, a pineal hormone, because of its wide activity spectrum, is a subject of much current interest for biologists and physicians. It has been demonstrated that pineal gland is not an exclusive source of melatonin synthesis. Melatonin synthesis has been found in different sites of the organism, and a major source of extrapineal melatonin is the gastrointestinal tract. The role of melatonin in gastrointestinal functions is considered in the present review.