[Features of the immune proteasome expression in ascite Zajdela hepatoma after implantation into Brattleboro rats with the hereditary defect of arginine-vasopressin synthesis].

The expression of the total proteasome pool, immune proteasome subunits LMP2 and LMP7, TAP1 and TAP2 transporters, as well as RT1A molecule of MHC class I was investigated in the ascite Zajdela hepatoma at the 10th day after implantation into Brattleboro rats with the hereditary defect of hypothalamic arginine-vasopressin synthesis (AVP) and into WAG rats with normal AVP expression. In Zajdela hepatoma cells implanted into Brattleboro rats the 3-fold increase of the total proteasome pool and LMP2 level and 8-fold increase of the LMP7 level was detected by Western blotting as compared to those in WAG rats. Differences in the LMP2 and LMP7 expression suggest variations in their functions, namely the important role of LMP7 in anti-tumor immunity. The growth of Zajdela hepatoma in WAG rats was accompanied by the decreased level of total proteasome pool as well as immune proteasome expression as compared to those in Brattleboro rats during the regression of tumor. The analysis of TAP1 and TAP2 revealed the pronounced expression of these peptide transporters in Zajdela hepatoma cells implanted into Brattleboro and WAG rats. The expression level of RT1A molecule of MHC class I was increased 3 times in Zajdela hepatoma cells implanted into Brattleboro rats as compared to WAG rats. Moreover, flow cytometric analysis of CD4- and CD8-lymphocytes number in the spleen of Brattleboro and WAG rats was performed at the 10th day after implantation of Zajdela hepatoma. The increased number of CD4- and CD8-lymphocytes was observed in the spleen of Brattleboro as compared to WAG. The increased subpopulations of cytotoxic T-lymphocytes and T-helpers might promote the tumor regression in Brattleboro rats. The reduced populations of CD4- and CD8-lymphocytes in the spleen of WAG rats were accompanied by the splenomegaly and tumor progression. The data obtained suggest that AVP deficiency in Brattleboro rats leads to the increase of the immune proteasome and MHC class I expression in Zajdela hepatoma cells, resulting in tumor immunogenicity and its elimination by the adaptive immunity.

[Expression of the RT1A molecule of the class I major histocompatibility complex in a Walker 256 tumor after transplantation into Brattleboro rats with a genetic defect of arginine-vasopressin synthesis].

The dynamics of expression of the RT1A antigen of the class I major histocompatibility complex (MHC) in a Walker 256 tumor after its transplantation into Brattleboro rats with a genetic defect of Arginine-Vasopressin synthesis in the hypothalamus was studied. Expression of the RT1A antigen was detected by means of Western-blotting and flow cytometry in the tumor cells on the 14th-17th days after transplantation. In addition, a simultaneous increase in the portion of cells that express the RT1A antigen and in the level of its expression per cell was observed. It is presupposed that at a deficiency of Arginine-Vasopressin, a renewal of expression of the class I MHC antigens, which results in an increase of immunogenicity of this tumor and regression, occurs in the Walker 256 tumor in the Brattleboro rats.

[The role of serotonin in the immune system development and functioning during ontogenesis].

In this study, we investigated the influence of serotonin on the development and functioning of T- and B-cell-mediated immunity during ontogenesis using the pharmacological model of serotonin depletion in rat fetuses. It has been demonstrated that prenatal serotonin deficiency resulted in a decrease in thymus and spleen weights, changes in their cellular composition, and long-lasting disturbances in cell-mediated and humoral immunity in postnatal ontogenesis. The data obtained suggest that serotonin may be considered a morphogenic factor in development of the immune system.

[Reparative regeneration of rat skin under influence of hollow cathode lamp (HCL) with manganese and copper line spectrum emission].

Influence of local light exposure by hollow cathode lamp with typical manganese and copper (HCL-Mn, Cu) line emission spectrum on posttraumatic regeneration rate of rat skin has been investigated. We performed the comparative analysis of the morphology and the differentiation ability of rat skin on the 15th and 24th days after full-thickness skin wound had been inflicted on rat dorsums. On the 15th day after injury, the experimental group (daily 30 s exposure for two weeks) showed scab loss, re-epithelialization, and hair regrowth, in contrast to the control rats, where scabs were still observed on the 24th day. Histological analysis revealed that in contrast to the control group the treatment with HCL-Mn, Cu resulted in the increased number of hair follicles and sebaceous glands, the decreased number of blood vessels and horizontal orientation of collagen fibers. The immunohistochemistry for OX-62 revealed that the number of dermal dendritic cells in the experimental groups was maximal on the 15th day, and then decreased to the 24th day after injury. The number of dermal dendritic cells was significantly lower in the control group. The immunohistochemistry for pan-keratins in the control animals revealed a high number of cells expressing different types of keratins, distributed in the main part of the epidermis on the 15th day after surgery, whereas in the experimental group the number of such cells was significantly lower and the cells were concentrated more close to the external part of the epidermis. The number of cells stained for keratin 19 was higher in the experimental group on the 15th day after surgery, whereas this number decreased in this group on the 24th day after surgery as compared to the control group. Thus, typical manganese and copper line spectrum emission emitted by hollow cathode lamp stimulates innate immunity, accelerates restoration of derma, skin epithelium and other skin derivates, and stimulates wound healing in general.

[Immune proteasomes in the developing rat spleen].

Changes in the structure of the rat spleen and the distribution of immune proteasomes in it during early postnatal development have been studied using double immunofluorescent staining of tissue sections with antibodies to the LMP7 immune proteasome subunit and to specific markers of T and B lymphocytes. It has been shown that the white pulp on postnatal day 5 is not yet colonized by lymphocytes and contains a smaller amount of immune proteasomes than the red pulp. At this stage, T and B lymphocytes concentrate mainly in the red pulp. On day 8, B lymphocytes occupy the marginal zone, while T lymphocytes aggregate into dense strands close to the white pulp. By day 18, T lymphocytes form periarteriolar sheaths in the white pulp, and the contents of immune proteasomes in the red and white pulp become equally high. An increase in the total content of immune proteasomes in the spleen on the third postnatal week was revealed in our previous study by Western blotting. In addition to T and B lymphocytes, immune proteasomes have also been revealed in other spleen cell types, probably in macrophages and reticular cells of the white pulp. Thus, the postnatal development of the spleen is associated with an increase in the contents of immune proteasomes in it.

The brain is one of the most important sources of dopamine in the systemic circulation in the perinatal period of ontogenesis in rats.

This study was designed to test the authors' hypothesis that dopamine passes from dopamine-synthesizing cells in the brain to the systemic circulation prior to the formation of the blood-brain barrier during ontogenesis. High-performance liquid chromatography studies demonstrated that peripheral blood dopamine levels before formation of the blood-brain barrier-in rat fetuses and neonates-are significantly higher than after formation of the barrier in adult rats, providing indirect evidence in support of the hypothesis. Furthermore, formation of the blood-brain barrier is accompanied by a significant increase in dopamine levels in the rat brain. Direct evidence for the hypothesis was obtained in the form of a sharp decrease in blood dopamine levels in fetuses after lesioning of dopamine-synthesizing neurons in the brain by encephalectomy.

Tactivin in the regulation of dexamethasone-induced apoptosis in thymocytes.

The level of natural apoptosis in rat thymus on day 18 of embryo development attained 25%, while at subsequent terms it was about 5%. In the spleen, this parameter gradually decreased from 15 to 37% starting from day 18 of embryo development to postnatal day 30. Tactivin prevented the development of dexamethasone-induced apoptosis in thymocytes of 30-day-old rats, but had no effect on spontaneous apoptosis. Tactivin can be used as a modulator of apoptotic processes.

[Dynamics of apoptosis and proliferation in the rat thymus and spleen during perinatal development].

The levels of spontaneous apoptosis and proliferation of the rat thymic and spleen cells, as well as their regulation by the hypothalamo-hypophysial system were studied during perinatal development. The apoptotic and proliferating cells in the thymus and spleen were assayed using flow cytometry with the DNA-specific dye propidium iodide. The level of apoptosis in the thymus reached 25% on day 18 of embryogenesis (E 18) and decreased to 5% thereafter. In the spleen, the level of apoptosis gradually increased from 15 to 37% during the period of E18 to day 30 of postnatal development (P30). The level of dividing cells in the thymus was 20-25% at all developmental stages studied. In the spleen, it was at a maximum on E18 (32%) and decreased almost twice on E21 (17%). On P7, the amount of proliferating cells again increased to 22% and then gradually decreased to 7% by P30. The surgical ablation of hypothalamus in utero on E18 did not affect cell apoptosis or proliferation in the thymus and spleen. The surgical ablation of both hypothalamus and pituitary led a twofold decrease of the level of apoptosis in the spleen and insignificant increase of the level of proliferation in the thymus. Thus, the numbers of cells in the embryonic thymus is regulated not only by the thymus itself, but also by the hypothalamo-hypophysial system. The programmed cell death in the embryonic spleen appears to be regulated by the hypothalamo-hypophysial system as well.

[The brain is one of the most important sources of dopamine in general circulation during perinatal ontogenesis in rats].

This study was aimed to test our hypothesis that dopamine synthesized in the neurons of the brain is delivered to the general circulation in rats during prenatal and early postnatal periods, i.e. before the establishment of the blood-brain barrier. Using the high performance liquid chromatography, it was demonstrated that the dopamine concentration and content in the peripheral blood in fetuses and neonatal rats (i.e. before the establishment of the blood-brain barrier) greatly exceeded those in adult rats. Moreover, the establishment of the blood-brain barrier was accompanied by the significant increase of the dopamine concentration in the brain. A drop of the dopamine concentration in fetal plasma after the microsurgical lesion of the forebrain and mesencephalon (encephalectomy) are considered as direct evidence in favour of our hypothesis.

Dopamine synthesis by non-dopaminergic neurons in the arcuate nucleus of rat fetuses.

The aim of the present work was to verify the hypothesis that non-dopaminergic neurons expressing individual complementary dopamine synthesis enzymes can perform the co-located synthesis of dopamine. According to this hypothesis, neurons expressing tyrosine hydroxylase use L-tyrosine for the synthesis of L-dihydroxyphenylalanine (L-DOPA), which then enters neurons expressing aromatic amino acid decarboxylase, which converts L-DOPA to dopamine. Experiments were performed using the mediobasal hypothalamus of rat fetuses, which mostly contains single-enzyme neurons (>99%) and occasional double-enzyme neurons (<1%). Controls were obtained from the fetal substantia nigra, which is enriched with dopaminergic neurons. High-performance liquid chromatography was used to measure levels of dopamine and L-DOPA in cell extracts and the incubation medium after incubation in the presence and absence of exogenous L-tyrosine. Addition of L-tyrosine to the medium led to increases in the level of synthesis and release of L-DOPA in the mediobasal hypothalamus and substantia nigra. In addition, L-tyrosine increased dopamine synthesis in the substantia nigra and decreased dopamine synthesis in the mediobasal hypothalamus. This regional difference in levels of dopamine synthesis is probably due to inhibition of the uptake of L-DOPA from the intercellular medium by neurons in the mediobasal hypothalamus containing aromatic amino acid decarboxylase, due to the competitive binding of the L-DOPA transporter by L-tyrosine. Thus, these results provide the first evidence for the co-located synthesis of dopamine by non-dopaminergic neurons expressing single complementary enzymes involved in the synthesis of this neurotransmitter.

[Ultrasound in prevention of postoperative immunodeficiency].

Phonoprophylaxis of postoperative immunodeficiency was developed and tested in 34 patients. From the first day of hospitalization to the day of elective surgery the patients were daily exposed to ultrasound on the area of the manubrium sterni according to the labile technique through petrolatum or glycerol medium (0.05 W/cm2, 120-150 s, each other day, 4-6 procedures per course). Immune status studies after surgery prove a prophylactic effect of the above ultrasonic procedures.

Non-dopaminergic neurons expressing dopamine synthesis enzymes: differentiation and functional significance.

The development and functional significance of neurons in the arcuate nucleus expressing tyrosine hydroxylase and/or aromatic L-amino acid decarboxylase were studied in rat fetuses, neonates, and adults using immunocytochemical (single and double immunolabeling of tyrosine hydroxylase and aromatic L-amino acid decarboxylase) methods with a confocal microscope and computerized image analysis, HPLC with electrochemical detection, and radioimmunological analysis. Single-enzyme neurons containing tyrosine hydroxylase were first seen on day 18 of embryonic development in the ventrolateral part of the arcuate nucleus. Neurons expressing only aromatic L-amino acid decarboxylase or both enzymes of the dopamine synthesis pathway were first seen on day 20 of embryonic development, in the dorsomedial part of the nucleus. On days 20-21 of embryonic development, dopaminergic (containing both enzymes) neurons amounted to less than 1% of all neurons expressing tyrosine hydroxylase and/or aromatic L-amino acid decarboxylase. Nonetheless, in the ex vivo arcuate nucleus and in primary neuron cultures from this structure, there were relatively high leveLs of dopamine and L-dihydroxyphenylalanine (L-DOPA), and these substances were secreted spontaneously and in response to stimulation. In addition. dopamine levels in the arcuate nucleus in fetuses were sufficient to support the inhibitory regulation of prolactin secretion by the hypophysis, which is typical of adult animals. During development, the proportion of dopaminergic neurons increased, reaching 38% in adult rats. Specialized contacts between single-enzyme tyrosine hydroxylase-containing and aromatic L-amino acid decarboxylase-containing neurons were present by day 21 of embryonic development; these were probably involved in transporting L-DOPA from the former neurons to the latter. It was also demonstrated that the axons of single-enzyme decarboxylase-containing neurons projected into the median eminence, supporting the secretion of dopamine into the hypophyseal portal circulation. Thus, dopamine is probably synthesized in the arcuate nucleus not only by dopaminergic neurons, but also by neurons expressing only tyrosine hydroxylase or aromatic L-amino acid decarboxylase.

[Tyrosine hydroxylase expression in differentiating neurons of the rat arcuate nucleus: inhibitory effect of serotoninergic afferents]. / Ekspressiia tirozingidroksilazy v differentsiruiushchikhsia neironakh arkuatnogo iadra krys: ingibitornoe vliianie serotoninergicheskikh afferentov.

In vivo studies, serotonine synthesis in the rat fetal brain was inhibited by p-chlorphenylalanine from the 11th to the 20th embryonic day. Serotonine depletion significantly decreased thyrosine hydroxylase content in the neurones of males and females on the 21st embryonic day and in males--on the 35th postnatal day. In vitro, a co-culture of arquate nucleus' and raphe nucleus' embryonic neurones resulted in a sex-specific increase of the thyrosine hydroxylase level in the former neurones. The raphe nucleus' neurones manifested an increased level of serotonine. The findings suggest an activating long-lasting effect of serotonine afferents on the thyrosine hydroxylase expression in differentiating neurones of the arquate nucleus in rats during prenatal ontogenesis.

[Non-dopaminergic neurons expressing dopamine synthesis enzymes: differentiation and functional importance]. / Nedofaminergicheskie neirony, ékspressiruiushchie fermenty sinteza dofamina: differentsirovka i funktsional'noe znachenie.

The study has evaluated in vivo, ex vivo and in vitro ontogenesis and functional significance of the arcuate nucleus neurons expressing either individual enzymes of dopamine synthesis, tyrosine hydroxylase or aromatic L-amino acid decarboxylase as well as both of them (dopaminergic neurons) in rats from the 17th embryonic day to adulthood. Monoenzymatic tyrosine hydroxylase-containing neurons were initially observed on the 18th embryonic day. On the 20-21 day, the monoenzymatic tyrosine hydroxylase- or aromatic L-amino acid decarboxylase-expressing neurons comprised more than 99% of the whole neuron population expressing the dopamine-synthesizing enzymes. The dopamine production in the fetus arcuate nucleus was sufficient to provide an inhibitory control of prolactin secretion like in adults. The data suggest a possibility of the dopamine synthesis in the fetus arcuate nucleus by the monoenzymatic neurons containing either tyrosine hydroxylase or aromatic L-amino acid decarboxylase-expressing neurons in co-operation.

[A clinical and experimental analysis of the effects of laser therapy]. / Kliniko-éksperimental'nyi analiz éffektov lazerterapii.

Thymus area was exposed to laser radiation in 180 Wistar male rats. Intact animals experienced a stress response to a single laser procedure (a rise in the levels of hydrocortisone, a fall in those of insulin, triiodothyronine and thyroxine). Longer exposure to laser radiation caused a break of the hydrocortisone mechanism. Similar effects were seen in the course exposure. The immune system was unchanged. In adjuvant arthritis laser radiation induced similar hormonal shifts and additional suppression of immunity. In 82 children with active juvenile rheumatoid arthritis laser therapy produced an insignificant effect, while in adults with moderate rheumatoid arthritis the response was good.

[The formation and regulation of the lactotrophic function of the hypophysis in the prenatal period of rat development. I. Prolactin secretion by the hypophysis and the role of dopamine in this process]. / Stanovlenie i reguliatsiia laktotrofnoi funktsii gipofiza v prenatal'nom periode razvitiia krys. I. Sekretsiia prolaktina gipofizom i rol' dofamina v étom protsesse.

Using radioimmunologic assay, we have studied the content of prolactin in the pituitary and its release into general circulation in 18-, 20-, and 22-day-old rat fetuses under normal conditions and after pharmacological block of dopamine receptors. Prolactin was found in the pituitaries of the fetuses from day 5 and in blood serum, from day 18; its levels were progressively increasing up to the end of prenatal development. Administration of haloperidol, an inhibitor of dopamine D2 receptors, to pregnant female increased the level of prolactin in fetus plasma from day 20 and diminished its content in the pituitary gland from day 22. These data provide evidence for secretion of prolactin by the pituitary and sensitivity of lactotrophs to dopamine during prenatal development.