[The study of interaction of different forms of human recombinant anti-mullerian hormone with a chimeric analogue of the AMH type II]. / Issledovanie vzaimodeistviia razlichnykh form rekombinantnogo antimiullerova gormona cheloveka s khimernym analogom ego retseptora II tipa.

The homodimeric glycoprotein, anti-mullerian hormone (AMH), described over 70 years ago by A. Jost, is the least studied member of the transforming growth factor beta superfamily. Despite the antitumor activity of AMH discovered at the end of the last century, the creation of effective drugs based on AMH is hindered primarily by the lack of information on the mechanism of various AMH forms interaction with a specific type II receptor (MISRII). Previously, we have shown that not only the full-length activated hormone but also its C-terminal fragment (C-rAMH) could bind to MISRII. In this work, using the surface plasmon resonance technique, we compared the interaction of three forms of recombinant AMH (rAMH) with the MISRII analogue - the chimeric protein MISRII-Fc containing AMH type II receptor and a Fc-fragment of the human IgG1 heavy chain. Comparison of the binding of MISRII-Fc, immobilized on a chip with group specificity for human immunoglobulins, to C-rAMH, to intact rAMH (pro-rAMH), and to rAMH containing one uncleaved monomer (hc-rAMH), showed that the KD of the complexes increased: 1.7 nM, 88 nM and 110 nM, respectively. Thus, we have shown that C-terminal fragment of AMH has the maximum affinity for the recombinant MISRII analogue, which indicates the prospects for the development of drugs based on this hormone derivative.

[Mullerian inhibiting substance type II receptor as a potential target for antineoplastic therapy]. / Retseptor antimiullerova gormona II tipa kak potentsial'naia mishen' dlia protivoopukholevoi terapii.

The review considers properties of the type II anti-Mullerian hormone receptor (mullerian inhibiting substance receptor type II, MISRII), a transmembrane sensor with its own serine/threonine protein kinase activity, triggering apoptosis of the Mullerian ducts in mammalian embryogenesis and providing formation of the male type reproductive system. According to recent data, MISRII overexpression in the postnatal period is found in cells of a number of ovarian, mammary gland, and prostate tumors, and anti-Mullerian hormone (AMH) has a pro-apoptotic effect on MISRII-positive tumor cells. This fact makes MISRII a potential target for targeted anti-cancer therapy. Treatment based on targeting MISRII seems to be a much more effective alternative to the traditional one and will significantly reduce the drug dose. However, the mechanism of MISRII-AMH interaction is still poorly understood, so the development of new anticancer drugs is complicated. The review analyzes MISRII molecular structure and expression levels in various tissues and cell lines, as well as current understanding of the AMH binding mechanisms and data on the possibility of using MISRII as a target for the action of AMH-based antineoplastic drugs.

[The dynamics of human total and activated anti-mullerian hormone serum levels in different life periods.]

Here, changes in the serum level of total anti-mullerian hormone (AMH) and its activated form in children of both sexes and women with different reproductive status are investigated. This TGFß superfamily cytokine is known to provide the formation of the male-type reproductive system in mammalian embryogenesis, and regulate folliculogenesis, spermatogenesis and the balance of sex hormones after birth. The biologically active form of the hormone (aAMH) is formed as a result of limited proteolysis of the AMH molecule; it is not reliably known in which tissues and under the action of which enzyme it occurs. The serum level of aAMH seems to be a more informative clinical indicator than the content of total AMH (tAMH), but there are no ELISA systems at the world market that provide direct quantitative detection of aAMH. In this work, quantitative detection of total hormone (tAMH) and its biologically active form (aAMH) in serum was performed using specially developed enzyme immunoassay systems. We showed that in girls, the total serum AMH level, as well as the concentration ratio aAMH / tAMH, practically does not change with age, whereas in boys, there is not only a significant decrease in the total serum AMH level previously described in the literature (Pearson correlation coefficient R = - 0.86, p <0.001), but also in the ratios of the aAMH / tAMH level (R = -0.531, p <0.001). It was also found that in pregnant women, the amount of total AMH and the proportion of aAMH in serum was significantly higher (p <0.01 and p <0.001, respectively) than in the control group women. The obtained results are in good agreement with the available data on the total and activated AMH content in the blood serum of people of different sex and age and indicate a change in the ratio of aAMH / tAMH serum levels in pregnancy. These data may be important both for deepening the understanding of AMH biology and for interpreting the results obtained using AMH detection based diagnostics.

[EXPERIMENTAL ALLERGIC ENCEPHALOMYELITIS IN ADULT RATS AFTER ADMINISTRATION OF INTERLEUKIN-1beta DURING DIFFERENT PERIODS OF EARLY LIFE].

Multiple sclerosis is a chronic disease of the CNS that affects people of working age, in which the targets of aggressive immune cells become the myelin and myeline producing cells, as well as neurons. It is assumed that a predisposition to MS is forming in childhood, due to common infections. In this paper the experimental allergic encephalomyelitis (EAE) was examined in rats administered IL-1beta at different periods of the early postnatal ontogenesis. EAE was induced in rats at the age of 3 months by single subcutaneous immunization with a homologous homogenate of spinal cord in complete Freund's adjuvant. The number of sick animals were evaluated, as well as the severity of the disease and its duration. It was shown that in rats after administration of IL-1beta on 1st and on 4th week of life EAE is more severe than corresponding control groups of rats. Discusses the damaging or protective effects of injections of IL-1beta during different periods of early postnatal ontogenesis, role of stress reactivity and communication with the "hygiene hypothesis".

[THE EFFECTS OF CHRO- NIC ADMINISTRATION OF MODERATE DOSE OF PROINFLAMMATORY CYTOKINE INTERLEUKIN-1ß ON RAT INVESTIGATIVE BEHAVIOR AND SPATIAL MEMORY].

The aim of this study was to discover the effects of chronic intraperitoneal administration of proinflammatory cytokine interleukin-1ß (IL-1ß) on rat investigative behavior and spatial memory. Rats were injected with a moderate pyrogenic dose of IL-1ß (0.5 mkg/kg) daily during 14 days (7 days before tests and 7 testing days). The behavior was examined in 23.5 hours after the previous injection of cytokine. The test battery included: "The open field" (within 3 consecutive days), "The exploration of novel objects", and "The Morris water maze". The animals treated with IL-1ß differed from the control animals in an essential decrease of locomotor activity, slight increase of anxiety and suppression of exploratory behavior. The impairment of spatial memory was not revealed.

The role of reactive oxygen species in membrane potential changes in macrophages and astrocytes.

Involvement of reactive oxygen species (ROS) in changes of the plasma membrane potential of mouse peritoneal macrophages and astrocytes (U118 cell line) under the action of different agents has been studied. Membrane potential was measured using the voltage-dependent fluorescent oxonol dye DiBAC4(3). Agonists which stimulate macrophages to release ROS (the fMLP peptide and platelet activating factor) caused prolonged hyperpolarization. Experiments with the fluorescent probe 2',7'-dichlorofluorescein diacetate have shown that astrocytes release ROS upon the action of C5a complement anaphylatoxin (but not C3a). The effect of C5a was accompanied with hyperpolarization of the astrocyte plasma membrane. Treatment of the cells with agents which do not induce ROS generation (C3a, lipopolysaccharide, interferon-gamma) depolarized the plasma membrane. Hyperpolarization of both cell types was significantly decreased in the presence of superoxide dismutase (but not catalase). Moreover, the O2- -generating system caused a marked hyperpolarization of both cell types. The data obtained suggest that O2- is involved in the macrophage and astrocyte hyperpolarization response.

Expression of cytokines by human astrocytomas following stimulation by C3a and C5a anaphylatoxins: specific increase in interleukin-6 mRNA expression.

C3a and C5a anaphylatoxins are two proinflammatory peptides generated during complement activation that act through distinct Gi protein-coupled receptors named C3aR and C5aR, respectively. We have demonstrated previously that human astrocytes expressed C3aR and C5aR constitutively and were able to produce a functional complement. In this study, we examined the effect of an anaphylatoxin stimulation on cytokine expression by human astrocyte cell lines. Interleukin (IL)-1beta, IL-6, tumor necrosis factor-alpha, and transforming growth factor-beta mRNA expression was studied by quantitative RT-PCR. Whereas IL-1beta, tumor necrosis factor-alpha, and transforming growth factor-beta mRNA levels remained unchanged, stimulation of astrocytoma cells (T98G, CB193, U118MG) by C3a, C5a, and peptidic C3aR and C5aR agonists induced an increase in the IL-6 mRNA level. The amount of IL-6 was markedly increased at 3 and 6 h and returned to the basal level at 9 h of stimulation. This response was specific, because pretreatment of cells with pertussis toxin or with polyclonal anti-C3aR or anti-C5aR antibodies completely blocked the IL-6 mRNA increase. The IL-6 response was also investigated at the protein level, but IL-6 protein was detected neither in cell lysates nor in supernatants of stimulated cells. The anaphylatoxin-mediated transcriptional activation of IL-6 gene suggests that C3a and C5a could play a role in priming glial cells during the inflammatory process in the brain.

Expression of complement components of the alternative pathway by glioma cell lines.

Glioma cell lines express proteins of the complement alternative pathway, namely C3, factor B, factor H, and factor I. Secretion of these proteins was shown by a sensitive and specific ELISA. C3 and factor H were rapidly secreted by glioma cell line CB193 and reached a concentration of 140 ng/ml/10(6) cells after 72 h of culture. Factor B and factor I were secreted at a lower rate and reached concentrations of 25 and 15 ng/ml/10(6) cells, respectively. Western blot and immunoprecipitation experiments showed that secreted proteins were identical to the corresponding plasma proteins. For factor H, besides the well known 150-kDa species, an additional polypeptide of 45 kDa with factor H immunoreactivity was observed. This species corresponded to the N-terminal truncated form found in plasma. In preliminary experiments, we observed control of these syntheses by cytokines. IL-1 beta significantly increased C3 secretion, with no effect on factor H. Secretion of factor H was enhanced by IFN-gamma. These results show that a glioma cell line could be a useful tool to study complement biosynthesis by glial cells in humans.