M. Pospíšil

Effects of D2-dopamine and α-adrenoceptor antagonists in stress induced changes on immune responsiveness of mice

The involvement of catecholamine receptors (alpha-adrenergic, D2-dopamine (DA)) was investigated in restraint stress influenced immune responses with concomitant changes of G-protein signal transduction. Impairment of the spleen morphology, TH1/TH2 cytokine network and natural killer (NK) cell function was observed. In vivo administration of specific antagonists prior to restraint stress reversed the immunosuppression. These findings demonstrate that D2-type dopaminergic mechanism represents the dominant component in regulation of Galphas/Galphai(1,2)/Galphaq/11-protein signal transduction and contribute to cell responses at postreceptor level of both, central nervous and immune systems. G-protein-coupled receptors (GPCRs) can modulate cytokine production and may play a regulatory role in immune effector mechanisms.

Glycodendrimeric ligands of C-type lectin receptors as therapeutic agents in experimental cancer

The increased knowledge about the receptor-ligand relationships of the cytotoxic effector cells is suggesting modalities for new systems of immunomodulation. The lectin receptors on natural killer cells (NK) and their recognition of carbohydrate ligands are part of these new perspectives.

Clustered ergot alkaloids modulate cell-mediated cytotoxicity

Dimers of agroclavine (1) and terguride (2), as well as a series of terguride oligomers, for example trimers (5, 6), tetramer (7), hexamer (8) and functionalized tergurides for further complex clustering were synthesized. Terguride oligomers were screened for their direct cellular toxicity on lymphoma cell lines in vitro and for their immunomodulating activities, represented by the natural killer (NK) cell-mediated cytotoxicity, as the most sensitive screening marker during immune responses. Dimers linked via aromatic spacer showed a high toxicity (1 microM) to lymphoma cells, which was not detected in other derivatives. In vitro and ex vivo experiments performed on mouse spleen lymphocytes in the presence of terguride oligomers demonstrated an immunosuppressive effect of dimers with aromatic spacer (4c-d) and NK cell stimulatory effect of terguride hexamer (8) and trimer with aliphatic spacer (5c). There is a considerable evidence that indolic part of molecule contributes to immunosuppressive action of terguride, which is potentiated in dimers carrying aromatic linker. This effect can be reversed by higher oligomerization of the respective alkaloids.

Particulate 1,3-β-d-glucan, carboxymethylglucan and sulfoethylglucan: Influence of their oral or intraperitoneal administration on immunological respondence of mice

The effect of orally or intraperitoneally administered particulate 1,3-β-d-glucan (PBG), carboxymethylglucan (CMG) or sulfoethylglucan (SEG), obtained from the culture filtrate ofSaccharomyces cerevisiae, on the functions of murine peritoneal adherent cells (PC) (peroxidase activity, nitric oxide synthesis), on relative organ mass and on proliferation of splenocytes was determined. The modulating activities after parenteral and non-parenteral administration of these polysaccharides were compared. Significant enhancement of NO production was observed only afterin vitro cultivation of PC in the presence of lipopolysaccharide (LPS) in groups of mice treated repeatedly orally with CMG, PBG and SEG at a dose of 50 mg/kg body mass. Peroxidase activity increased significantly after repeated oral administration of CMG and PBG at doses 150 and 50 mg/kg, SEG 150 mg/kg body mass. The peroxidase activity and NO synthesis in mice given a single intraperitoneal injection of glucans (15 mg/kg body mass) were slightly higher than those after oral administration. Neither a significant enhancement of relative organ mass nor enhancement of the proliferative response of splenocytes toin vitro added stimuli (LPS, phytohemagglutinin) after repeated oral or single intraperitoneal administration of β-glucans was observed.

Phenol—water extracts of gram-positivelisteria monocy togenes and gram-negativesalmonella typhimurium. comparison of biological activities

Using phenol—water extraction, a lipopeptidopolysacoharide complex (LPPS) was isolated fromListeria monocytogenes. Some biological and immunological properties of LPPS were compared with lipopolysaccharide isolated by the same procedure fromSalmonella typhimurium. LPPS possesses low pyrogenicity, but the immunological activity is comparable with LPS: slightly lower adjuvant and polyolonal stimulating effect, almost equal mitogenic effect on mouse spleen cells and higher mitogenic effect oh human peripheral blood cells, The results are discussed in connection with the chemical structure of both substances.

Peripheral membrane molecules of leukocytes and NK cytotoxicity.

Some leukocyte effector cell-surface molecules movement toward the adjoining target cells takes place during the reaction of NK cytotoxicity (NK R). The majority of the moving molecules are usually anchoredvia a divalent-ion-dependent interaction (PMM-M2+). The released PMM-M2+ can interact also with the secreted tumor necrosis factor alfa (TNF-α). In agreement with PMM-M2+ movement, the number of TNF-α binding sites on the target cell surface increases during NK R. In addition, antibodies against PMM-M2+, as well asd-mannose- or N-acetyl-d-glucosamine-terminated oligosaccharides of PMM-M2+ inhibit NK R. A more detailed analysis of PMM-M2+ with monoclonal antibodies used flow cytometry and cell-surface biotinylation. Only 3 of 31 tested CD antigens (CD2, LAK-1 and CD45) were passed through this first strongly restricted experimental screening. The EDTA-released LAK-1 antigen, but not CD2 and CD45, interact with TNF-α and cell surfacevia a mannose-inhibitable interaction dependent on the presence of Ca2+ ions. The mechanism of possible participation of PMM-M2+ in cytotoxic events is discussed in relation to Ca2+ influx and subsequent cytolysin secretion.

Characterization of antigen-specific receptors from pig lymphocytes

Antigen-specific receptors were isolated from the surface of spleen and lymph node cells of ARS-BGG-stimulated miniature pigs using an immunoadsorption technique. The yields of the receptors can be increased by 20-hr cultivation of the cells protein-free medium prior to the isolation procedure. In this case antigen-specific molecules can be isolated not only from the surface of the cells but also from the culture medium. 125I-labelled isolated receptors were separated by SDS-PAGE into six fractions. The first corresponded to aggregated molecules and IgG, the mol. wts of the other fractions were 56,000, 35,000, 26,000 and 10,000. It has been shown that they all were able to bind antigen but only the first two reacted with anti-Ig antibodies and Staphylococcus aureus protein A-Sepharose 4B. Gel filtration of the same sample of 125I receptors on Sephacryl S-200 revealed the presence of four peaks. Three of them were able to bind antigen as detected by radioimmunoassay. Fractions of all three peaks reacted also with anti-Id and anti-R (anti-Ig- receptors) antisera, but only fractions of the first two peaks reacted with anti-Ig antisera. The finding that Ig- receptors and more heterogeneous than Ig+ receptors is discussed. No reaction of isolated 125I receptors with anti-SLA and anti-SLB antisera used was detected in the solid-phase radioimmunoassay.

Ergot Alkaloid-Induced Cell Proliferation, Cytotoxicity, and Lymphokine Production

On the basis of the structural similarities among ergot alkaloids (EA)1 and important mediators such as noradrenaline, serotonin and dopamine, the interaction of EA with receptors for these mediators and wide range of their biological effects could be explained.2 From the functional point of view these effects are mediated by Q-adrenergic3,4, dopamine5 or serotonin receptors.6 Lymphoid cells possess membrane receptors for a variety of hormones that affect DNA synthesis and proliferation via the ubiquitous cAMP (cGMP) system.7,8 The process of hormone action often goes through cell receptors and membrane adenylcyclase to the cyclic nucleotides that function as transmitters of the hormonal message to the genome, thereby resulting in a change in the physiological status of the cell in question. Neurotransmitters may modulate immune functions, e.g., T cell differentiation, NK cell function6, T and B cell response to lectins, and macrophage functions.9 Corresponding receptors to neurotransmitters have been described on mature and developing lymphocytes.10,11 Therefore, we have focused on determining the direct effects of EAs on some immune functions. We tested lymphocyte proliferative responses, cell-mediated cytotoxicity, and capability to produce cytokines.

Cultivation of mesenchymal tissue with reference to antibody formationin vitro

The optimal conditions for the cultivation of rabbit lymphocytes in suspension and antibody formation by these cellsin vitro were studied. The lymphocytes were cultivated in medium in which serum was replaced by serum albumin containing the alpha globulin fraction. Several media were used, with different amounts and proportions of amino acids, vitamins and glucose. The cells survived best in medium in which a component with a high molecular weight was present and in which the glutamine concentration was 0.5–1 mM and the amount of glucose was 0.4–0.5%. Among the other factors determined, the initial pH had a significant influence on survival of the tissue, the optimal limits being 6.9–7.2. Under these conditions 55–60% of the cells survived during 120 hours’ cultivation. The conditions and factors influencing these findings are discussed. Study of the formation of antibodies againstBrucella suis in vitro showed that antibody formation increased only up to 48 hours, after which it remained at the same level.AbstractИзучались оптимальные условия культивирования кроличьих лимфоцитов в суспензии и образование антител этими клетками in vitro. В культивационной среде сыворотку заменили сывороточным альбумином с примесью альфа-глобулиновой фракции. Было испытано несколько видов культивационной среды с различным количеством и содержанием аминокислот, витаминов и глюкозы. Лучше всего клетки выживали, если среда содержала высокомолекулярный компонент, если глутамин присутствовал в концент рации от 0,5 до 1 mM, а количество глюкозы колебалось от 0,4 до 0,5%. Из других изучавшихся факторов существенное влияние на выживание тканей оказывал исходный pH, в оптимальных пределах от 6,9 до 7,2. При этих условиях в течение 120-часовой культивации переживает 55–60% клеток. Исследования образования антител против Brucella suis in vitro показали, что повышение продукции антител наблюдается только до 48-го часа культивации, позднее же титр антител становится постоянным.—Обсуждаются условия и возможности воздействия на это явление.

Cultivation of lymphatic cells in protein-free medium and chemical study of the products

Rabbits immunized withBrucella suis within a period of one week formed antibodies in high titres. These antibodies were of a macroglobulin character only. Cells from the spleen and popliteal lymph nodes were cultivatedin vitro in protein-free medium, in which the only macromolecular substance was dextran or carbowax.Physico-chemical analysis of secretion products was not succesful, since under the given cultivation conditions the cells started to disintegrate from the outset and the medium contained nucleic acids as well as proteins. Some of these substances were present in particles of the size of different subcellular particles. After cultivation of spleen and lymph node cells, antibodies were detected by an agglutination test in media containing dextran and carbowax. When particles substantially larger than the antibody molecule were removed by ultracentrifugation, the agglutination antibody titre in the medium fell.AbstractУ кроликов, после иммунизации Brucella suis в течение недели наблюдались высокие титры антител. Все эти антитела обладали свойствами макроглобулинов. Клетки селезенки и подколенных лимфатических узлов этих кроликов культивировали в безбелковой среде, содержавшей в качестве единственного высокомолекулярного вещества декстран или карбовакс.Анализ продуктов секреции этих клеток физико-химическими методами не удавался, так как при культивации в этих условиях сразу же начинается распад клеток и среда содержит не только белки, но и нуклеиновые кислоты. Часть этих веществ присутствует в виде различных гранул с размерами порядка субклеточных частиц. Антитела в среде, в которой культивировали клетки селезенки и лимфатических узлов и которая содержала декстран или карбовакс, были обнаружены с помощью реакции агглютинации, причем титр антител в среде понижался в том случае, если из нее путем ультрацентрифугирования удаляли частицы, значительно превышавшие по своим размерам молекулы антител.