Analysis of the Healthy Subject Response to Prolonged Contact with Tuberculosis Patients.

Prolonged contact of healthy subjects with Mycobacterium tuberculosis can change their blood formula and immune status, thus reflecting adaptive reactions to constant antigenic load. The peripheral blood analysis of health care workers in a tuberculosis hospital demonstrates changes in cell populations which prevent development of tuberculosis, in particular, CD4+ Т cells and CD3+ Т cells. It is shown that the number of the memory CD4+ Т cells specific to M.tuberculosis antigens which produce interferon gamma depends on the duration of work contact with tuberculosis patients. The use of health care workers' blood characteristics as a control for tuberculosis patients is discussed.

[The Role of Extracellular Calcium Ions Reduction in T Cell Activation in Human Peripheral Blood].

Calcium plays a fundamental role in many essential live functions. It is well-known that many infections can lead to hypocalcaemia in humans. The importance of extracellular calcium concentration ([Ca2+]o) in T-cell activation is well recognized, but [Ca2+]o levels necessary for lymphocyte activation have not been investigated in detail. Whole blood from healthy donors was stimulated by immobilized anti-CD3 plus soluble co-stimulation anti-CD28 mAb and activation parameters have been analyzed. Interleukins-2 (IL-2), JL-4, IL-17A, interferon-gamma (IFN-γ) and tumor necrosis factor-alpha (TNF-α) production and CD4+CD69+, CD4+CD25+, CD4+(CYTOKINE)+ T cells were used as activation parameters. To change current [Ca2+]o values in PB we used EGTA. No EGTA influence on resting T cells was shown. Dependences of T-cell activation parameters on various [Ca2+]o were studied. These data indicate that extracellular Ca2+ chelation by EGTA leads to non-linear dependences of T-cell activation parameters on [Ca2+]o values. Obtained dependence of the number of CD4+CD69+ T cells from EGTA can be represented as a nonlinear curve with two distinct peaks. Cytokines production by activated Th1, Th2 and Th17 cells and CD4+(CYTOKINE)+ T cells have been analyzed in dependence on EGTA. The first peak is close to the norm-pathology border. The second peak is in the pathological [Ca2+]o values of PB. However, the local minimum between them is entirely in the "pathological" range. Obtained dependences can be represented as a nonlinear curve with two distinct peaks. All dependences shape have similar curve describing the influence of [EGTA] values on activated CD4+CD69+ T cells. Physiological significance of obtained data discussed.

[The Analysis for Probable Reasons of Cd4+ T-Cell Activation Non-Linear Dependence on Extra Cellular Calcium Ion Concentration in Human Peripheral Blood in vitro].

The analysis for probable reasons of CD4+ T-cell activation non-linear dependence on [Ca2+]o in HPB in vitro is the general aim of current work. At the beginning we pursued the analysis of receptor-dependent (the mixture of monoclonal antibodies (mAbs) to CD3 and CD28 molecules) and receptor-independent (phorbol-myristate-acetate and ionomycin mixture) means to activate T cells in vitro with different [Ca2+]o in HPB. The key role of intracellular T-cell signaling systems in activated T cells in their non-similar sensitivity to calcium ions in the blood was shown. The analysis of differentiation next stages of CD4+ T-cell activation in vitro relatively [Ca2+]o in PHB demonstrates the key role of the earliest induction stages in non-similar sensitivity to calcium ions in CD4+ T-cell activation in vitro. According to the pursued analysis; the non-similar sensitivity of CD4+ T-cell in vitro to activation is in no-way connected with pace differences on the primary stages of activation process. The comparison of CD4+ memory T cells with their naive T-cell precursors in the cell activation process in hypocalcemia conditions was made in the separate experimental series. The 1st maximum consists in average of 85% CD4+CD45R0high CD69+ memory T cells. Naive CD4+CD45RAlowCD69+ T cells constitute the remainder 15%. The 2nd maximum almost completely consists of CD4+CD45R0+CD69+ memory T cells. The ratio between CD4+CD69+ T cell maximums depends on donor ages and represents linear dependence with R = -0.981. The most probable candidate on the role of CD4+ T cell, being capable of activation in hypocalcemia conditions, are memory T lymphocytes, being resistant to ionomycin action (I R) subset. To check this assumption the mononuclear cells and their IR-fraction were prepared from donor PB. Then the mononuclear cells and their IR-fraction were activated by mAbs mixture at different [EGTA] values. For IR-fraction, enriched with CD4+CD45RA-CD45R0+ memory T cells, slightly seen 1st maximum and drastic 2nd maximum in the "pathology" [Ca2+] region was observed. Most likely, namely, at the 2nd maximum, there is IR CD4+CD45RA-CD45R0+ memory T cell majority, having changed intracellular calcium signaling system, to be activated. Hence, the existence for CD4+ T cell activation two maximums in hypocalcemia conditions is connected with the presence of two subsets of CD4+ memory T cells, differing in their calcium-dependent intracellular signaling system, in HPB.

Role of extracellular calcium ions at early stages of human T-lymphocyte polyclonal activation in vitro.

In this work we comparatively analyzed interleukin-2 (IL-2) and interferon gamma production (IFN-gamma) and also CD69 and CD25 expression by activated T-cells depending on extracellular calcium concentration ([Ca2+]e), which was varied with EGTA. The expression of CD69 molecules on the surface of T-cells depended only on the presence of phorbol myristate acetate, occurred at [Ca2+]e higher than 0.2 mM, and did not require the presence of ionomycin. The increase in [Ca2+]e by itself cannot induce expression of CD25 and CD69 molecules by activated cells. The values of [Ca2+]e, at which maximal fractions of CD3+CD69+(IL-2)+, CD3+CD69+(IFN-gamma)+, and CD3+CD25+ activated T-cells were reached, never coincided with mean values of [Ca2+]e for healthy donors and were different from each other. So, there is different [Ca2+]e dependence for initial stages of activated T-cells differentiation. The relation between T-cells activation parameters and their differentiation is discussed.

Calcium homeostasis change in CD4+ T lymphocytes from human peripheral blood during differentiation in vivo.

Resting naïve CD4+(CD45R0-)CD45RA+ T cells are sensitive to ionomycin. In contrast, resting CD4+(CD45RA-)CD45R0+ memory T cells show resistance to this Ca2+ ionophore. In the present study, the ability of activated T lymphocytes to respond to ionomycin during the transition from naïve precursors into memory T cells has been analyzed. Activated CD4+(CD45RA+)CD45R0+ T cells are always present both in human peripheral blood (HPB) and in the ionomycin-resistant (IR) fraction. Therefore, some activated T cells are resistant toward the Ca2+ ionophore. CD69 molecules are markers of the very early stage of T cell activation. However, CD4+CD69+ T cells have never been found in the IR fraction. Thus, the majority of CD4+ T lymphocytes at the early stage of activation are ionomycin-sensitive cells. The proportion of CD4+CD25+ T cells did not differ significantly in HPB and in the IR fraction. The presence of CD4+CD25+ T lymphocytes in the IR fraction reflects changes in the Ca2+-signaling pathway at this differentiation step of activated cells. Depending on the expression level of CD25 molecules, the population of CD4+CD25+ cells is divided in T-regulatory (CD25high) and proliferating (CD25low) subpopulations. The action of ionomycin results in a decrease in the portion of the CD4+CD25low T-cells, but it leads to an increase in the proportion of the CD4+CD25high T lymphocytes. Consequently, greater portion of CD4+CD25high T lymphocytes and smaller portion of CD4+CD25low T cells are IR cells. Expression of HLA-DR molecules can be used as the marker for the late activation step. The IR fraction is significantly rich in CD4+HLA-DR+ T lymphocytes in comparison to the blood of the same donor. The link between different differentiation steps of CD4+ T-lymphocytes and alterations in calcium ion homeostasis is discussed.

[Phenotype characteristic of the ionomycin-resistant CD4+ subset of the peripheral blood T-lymphocytes]. / Ionomitsin-rezistentnaia subpopuliatsiia CD4+ T-limfotsitov perifericheskoi krovi cheloveka. Fenotipicheskaia kharakteristika.

The differential sensitivity of peripheral blood (PB) CD4+ T lymphocytes to the calcium ionophore ionomycin was investigated. Effect of ionomycin exerted on T cells was time- and dose-dependent. We have shown that resistant cells belonged to some distinct T cell subsets. The resting naive CD4+CD45RA+ T cells showed a little, if any, resistance to ionomycin treatment. The primed CD4+CD45R0+ memory T cells behaved similarly as did ionomycin-resistant (IR) cells. Although IR CD4+ T cells had a typical "memory" phenotype, some quantitative differences were found in expression of CD11a, CD28, CD29, CD62L and CD243 markers between PB CD4+CD45R0+ T cells and corresponding IR cells.

Immunological memory in CBA and CBA/N mice after vaccination with Mycobacterium bovis (BCG).

In inbred CBA and CBA/N mice immunological memory was induced by subcutaneous injection of Mycobacterium bovis (BCG). Experiments with adoptive transfer of spleen T cells and ionomycin-resistant T cells (memory cells) between CBA and CBA/N mice in various combinations showed that immunological memory was not formed in CBA/N mice, but can be induced by adoptive transfer of cells from CBA mice.

Reduction of Ca2+-transporting systems in memory T cells.

Antigen-specific B and T lymphocytes make up the material grounds of immune memory, their main functional distinction from the so-called "naive" cells is due to the rapid and enhanced response to the antigen-pathogen. An essential distinction between the memory and naive T cells is different sensitivity of these two subpopulations of T lymphocytes to Ca2+-ionophores. Comparative analysis of Ca2+ responses of the immune memory T lymphocytes and naive T cells of mouse CBA/J line to the addition of Ca2+-mobilizing agents concanavalin A, thapsigargin, and ionomycin was carried out. These compounds in concentrations increasing [Ca2+]i in naive cells had no effect on [Ca2+]i in memory cells. Thus, the Ca2+ entrance into memory cells was not activated by exhaustion of intracellular resources. Estimation of intracellular resources of Ca2+, mobilized by ionomycin and thapsigargin in Ca2+ free medium has shown the absence in memory T cells of the intracellular Ca2+ pool, which may be one of factors of their resistance to ionophores. Reduction of the system of Ca2+ influx into memory T cells was shown using the SH-reagent thimerosal. Memory T cells appear to be resistant to "Ca2+ -paradox." Their incubation with 0.5 mM EDTA in the presence or absence of Ca2+ -mobilizing compounds followed by addition of 2 mM CaCl2 did not result in induction of Ca2+ influx into these cells.

[Induction of anti-meningitis immunity using the synthetic peptides. I. The immunoreactive synthetic fragments of porin A from Neisseria meningitidis]. / Induktsiia protivomeningitnogo immuniteta s pomoshch'iu sinteticheskikhpeptidov. I. Immunoaktivnye sinteticheskie fragmenty belka porina A iz Neisseria meningitidis.

Fourteen peptides corresponding to sequences of all the exposed and some of the transmembrane protein regions of porin A from the outer membrane of Neisseria meningitidis strain B:15:P1.7,16 were synthesize. Mice of various lines were immunized with the free peptides not conjugated with any protein carrier. It was shown that the majority of the peptides possess immunogenic properties. Two peptides were identified binding to antibodies present in the serum of mice after meningitis. Protective properties of a number of the synthesized peptides were studied, and three peptide sequences inducing mice protection from an experimental infection with N. meningitidis were identified.

Muramyl peptide-binding sites are located inside target cells.

Using flow cytometry and fluorescence polarization analysis, specific muramyl peptide-binding sites were shown to be located inside T-lymphocytes, macrophages and neuroblastoma cells, but not inside B-cells. No binding sites were found on the cell surface. The number of binding sites for each cell type was determined. Two types of binding sites were observed for myelomonocytic WEHI-3 cells with Kd values of 21 and 540 nM. Inhibition analysis demonstrated that for effective binding, an intact glycopeptide molecule and D-configuration of isoglutamine residue are important.

[The effect of structural-chemical characteristics of water-soluble polyelectrolyte complexes of ovalbumin on their immunological properties]. / O vliianii strukturno-khimicheskikh osobennostei vodorastvorimykh poliélektrolitnykh kompleksov oval'bumina na ikh immunologicheskie svoistva.

Immunogenicity of soluble protein antigens in the complexes with synthetic polyions may be regarded as depending both on the nature of polymer carrier and the structure of the protein-polyelectrolyte complex. The immunogenicity of stable soluble complexes of ovalbumin (OA) with polycation - quaternized poly-4-vinylpyridine (C-1) and copolymer of acrylic acid and 2-methyl-5-vinylpyridine (C-2) have been evaluated. Immunization of mice by C-1 have induced a vigorous formation of the anti-OA IgG antibodies and IgE homocytotropic antibodies, while immunogenicity of OA in C-2 was comparable with that of OA alone. The analysis of the structural-chemical features of the complexes investigated has shown that enhanced immunogenicity of C-1 may be due to (1) the non-homogeneous distribution of protein globulae among polycation macromolecules and to (2) the formation of complex with an asymmetrical structure, to (3) the high ability of C-1 to adsorb on a surface of the lymphoid cells and to induce a formation of intercellular aggregates. An enhancing of a stability and a size of C-2 in the presence of Cu2+ shows no influence on a immunogenicity of OA. An immunogenicity of both types of complexes does not depend upon the access of determinants of OA to antibodies so far as it has been shown that complex formation in both cases are not accompanied by an alteration of antigenicity and allergenicity of OA.

[Two qualitatively different structuro-functional states of mitochondria]. / Dva kachestvenno razlichnykh strukturno-funktsional'nykh sostoiania mitokhondrii.

Low (120 mosM) tonicity of incubation media of mitochondria was found to be associated with anomalous phase transition at 19--26 degrees C. A rise in temperature caused a decrease in the pyrene excitation in border lipids of the mitochondrial membrane. Within this temperature range the quenching of intrinsic protein fluorescence by pyrene was sharply decreased. It may be inferred from these data that at 100mosM tonicity and temperatures below 19 degrees C, mitochondrial membrane proteins are in an aggregated state. At temperatures above phase transition protein deaggregation takes place. It was shown that a decrease in tonicity from 300 to 120 mosM at 15 degrees C or a rise in temperature from 15 degrees to 37 degrees C at 300 mosM tonicity increased the phosphorylation of the 52 kDa mitochondrial protein. It was assumed that swelling of mitochondria in hypotonic media simulates one of the steps of the hormone-induced signal transfer in mitochondria in vivo.

Microcalorimetry and electrophoresis of the erythrocyte membrane of spontaneously hypertensive rats.

Abnormalities of both structure and function have been described in the erythrocyte membrane of spontaneously hypertensive rats (SHR). In order to elucidate the molecular basis of these abnormalities we have carried out differential scanning microcalorimetry of the erythrocyte membrane and gel electrophoresis of membrane polypeptides. The partial enthalpy of so-called 'C-transition' (at 63 degrees C) was found to be increased. This may be explained by increased content of band 3 protein in SHR erythrocyte membrane.

[Physico-chemical properties of the erythrocyte membranes of normotensive and spontaneously hypertensive rats]. / Issledovanie fiziko-khimicheskikh svoistv membrany éritrotsitov normotenzivnykh i spontanno gipertenzivnykh krys.

To detect the substrate of molecular alterations in a plasma membrane, found earlier to be typical of chronic hypertension, microcalorimetric study of erythrocyte membranes of spontaneously hypertensive rats was performed. It was found that characteristics of some irreversible therminal transitions in the membranes differ from the corresponding control values. The supposed predominant role of membrane protein denaturation in these transitions is confirmed by the analysis of temperature-dependent infrared spectra, spectra of membrane protein fluorescence and circular dichroism.

[Various characteristics of erythrocyte membrane proteins in rats with spontaneous hypertension]. / Nekotorye osobennosti membrannykh belkov éritrotsitov krys so spontannoi gipertenziei.

Differential scanning microcalorimetry demonstrated an increase in specific heat capacity of erythrocyte membranes of spontaneously-hypertensive rats around 63 degrees C (C-transition). Disc electrophoresis with sodium dodecyl sulphate showed the percentage of band 3 peptides to be increased in relation to all membrane peptides in these membranes. It is suggested that the elevated content of band 3 peptides, which are known to participate in the formation of anion channels in mammalian erythrocyte membranes, may be one of the causes of increased specific heat of C-transition.

[Microcalorimetric study of erythrocyte membrane suspensions in normal subjects and in hypertension]. / Mikrokalorimetricheskoe issledovanie suspenzii membran éritrotsitov v norme i pri gipertonicheskoi bolezni.

Microcalorimetric method was used to study the temperature dependence of thermal absorption of human red cell membrane suspension in health and essential hypertension. In disease, the temperature dependence of individual irreversible thermoinduced transitions characteristic for human red cell membranes was appreciably changed, which was comparable with disorders of protein-lipid interactions discovered before by fluorescent examination, while elevated enthalpy of one of the transitions pointed to the possibility of enriching these membranes with band 3 protein. Unlike normal, red cell membranes in essential hypertension exhibited a special thermoinduced irreversible transition with the thermoabsorption maximum about 41 degrees C.

[Study of cation binding to myosin subfragment I using the fluorescent probe Eu3+]. / Izuchenie sviazyvaniia kationov s subfragmentom I miozina pri pomoshchi fluorestsentnogo zonda Eu3+.

It was shown that binding of the cation Eu3+ to myosin subfragment I (SI) results in fluorescence with a maximum at 595 nm which is increased as EuCl3 concentration rises. The ATPase activity of SI is simultaneously enhanced. An addition of bivalent cations (Ca2+ and Mg2+) causes quenching of fluorescence of the bound Eu3+ by 8-12%, which corresponds to Eu3+-ATPase inhibition by Mg2+. At low (down to 0.1 mM) concentrations of Eu3+ no fluorescence quenching by Mg2+ or Ca2+ takes place; under these conditions Mg2+ activate ATPase of SI in the presence of Eu3+. Eu3+ activate SI ATPase in the presence of low (down to 0.1 mM) concentrations of Ca2+, but exerts an inhibition action at high concentrations of Ca2+. NaCl does not affect the fluorescence intensity of bound Eu3+ but considerably inhibits the ATPase activity of SI in the presence of EuCl3. An existence of a bivalent cation binding site in the vicinity if the SI active center is postulated. Eu3+ whose ionic radius is close to that of Ca2+ interacts with protein surface and occupies this site as well, thus determining the activation of SI ATPase and can be replaced from it by Ca2+ and Mg2+, but not by Na+. Hence bivalent and monovalent cations are bound at different sites. The data obtained provide another proof in favour of a hypothesis suggesting that the regulation of myosin ATPase activity by bivalent and monovalent cations can be mediated by binding of these cations to the protein.

Evidence of altered structure of the erythrocyte membrane in spontaneously hypertensive rats.

1. The membrane structure of erythrocytes of rats with different forms of arterial hypertension was studied by means of two hydrophobic fluorescent probes (diphenylhexatriene and pyrene). 2. Microviscosity of hydrophobic areas of erythrocyte membrane of spontaneously hypertensive rats was found to be increased compared with that of membranes from normotensive control rats. 3. No alterations of membrane structure of erythrocytes of deoxycorticosterone-treated rats and renal hypertensive rats were found.

[Viscosity of free and protein-bound lipids in membranes]. / Izuchenie viazkosti svobodnykh i sviazannykh s belkom lipidov v membranakh.

Structural transitions induced by temperature in the lipid bilayer of rabbit sarcoplasmic reticulum membranes have been detected by means of the hydrophobic fluorescent probe pyren. Using the phenomenon of energy migration from membrane protein tryptophan residues to pyren a correct estimate of lipid viscosity near protein molecules was made by an empirical formula. The structural transitions of free and protein-bound lipids proceeded at the same temperature intervals. It was found that viscosity of protein-bound lipids was higher than that of free lipids, but they became equal at 36--39 degrees C.