Role of different B-cell subsets in the specific and polyclonal immune response to T-independent antigens type 2.

Role of different B-cell subsets in the immune response to T-independent antigen type 2 (TI-2) was studied. BALB/c and C57BL/6 mice were immunized by polyvinylpyrrolidon (PVP), and the numbers of antibody- and Ig-forming cells (AFC and IFC, respectively) were determined by ELISPOT method. The number of cells producing non-specific Ig (nIFC) was calculated as the difference between the number of IFC and AFC; the number of nIFC induced by PVP was calculated as the difference between the number of nIFC in immune and control splenocytes. Immunization by PVP induced not only the AFC appearance, but also the increase in the number of the antigen-induced nIFC. The treatment of splenocytes by anti-CD5 antibodies and guinea pig complement reduced the increase in the numbers of newly formed AFC and nIFC to approximately 40% of control level. It means that CD5+ cells play an important role not only in the specific, but also in polyclonal immune response to non-self TI-2. To be sure that the decrease of AFC and nIFC numbers is due to depletion for CD5+ B-cells, but not CD5+ T-cells, splenocytes were separated to B-1 and B-2 subsets, and the numbers of AFC, IFC and nIFC were determined in each B-cell subpopulation separately. The overwhelming majority of newly formed AFC and nIFC was detected in B-1 subset. The numbers of AFC and nIFC in B-1 compartment was approximately 10-fold greater than in B-2 cells. A close parallelism between AFC and nIFC formation was observed. It is concluded that specific and polyclonal immune response to non-self TI-2-PVP-depends mainly on CD5+ B-1 subset.

[Production of monoclonal antibodies to T-independent type 2 antigens with OptiMem-Iscove medium]. / Poluchenie monoklonal'nykh antitel k T-nezavisimym antigenam 2 tipa s ispol'zovaniem credy OptiMem-Iscove .

To obtain monoclonal antibodies to T-independent antigens of type 2, having low immunogenicity and incapable of inducing the appearance of memory cells, the use of the medium OptiMem-Iscove (1:1) with 10% of fetal serum, glutamine (50 mM) and antibiotics (100 units/ml) is proposed. The main advantage of this medium is the possibility of cloning cells without the use of feeders. The method has been approved in the process of obtaining monoclonal antibodies (McAb) to TI-2 antigens, both bacterial (S3) and synthetic (PVP), as well as to McAb to T-dependent antigen (alpha-chains of human IgA).

[Human monoclonal antibodies to synthetic viral peptides]. / Monoklonal'nye antitela cheloveka k sinteticheskim peptidam virusa.

Cells producing human antibodies to synthetic viral peptides MH-24 (corresponding to 302-325 residues of HIV-1 gp120) and p107 (main peptide of EBV antigen EBNA-1) were fused with human x mouse heteromyeloma B6B11. The efficacy of specific hybridization was 40%. Hybrid cells H1F2 (MN-specific) and HF4 (p107-specific) were cloned by the limiting dilutions method. Monoclonal antibodies (MAb) were tested in enzyme immunoassay on a panel of synthetic peptides. All resultant MAbs were polyspecific for tested synthetic peptides. The data permit a conclusion that previously detected polyspecificity of human cultural antibodies to virus peptides is due to the true polyspecificity of antigen-binding centers of MAb, but not to the presence of strange cells in the culture.

Role of different lymphocyte subpopulations in the formation of non-specific immunoglobulins induced by antigen injection.

The formation of antibody and non-specific immunoglobulin under the influence of T-dependent (TD) and type 2 T-independent (TI-2) antigens in mice of two congenic strains CBA (Lyb5-, Lyb5+) and CBA/N (Lyb5-) was studied. TD antigens induced in mice of both strains not only the appearance of antibody-forming cells (AFC), but also a great increase in the number of cells producing non-specific immunoglobulins (nIFC). TI-2 antigens induced the AFC and antigen-dependent nIFC formation in CBA mice only. It is concluded that during immune response to TI-2 antigens not only the AFC appearance but the increase in nIFC formation (polyclonal activation) is due mainly to the mature Lyb5+ B cells.

A new approach to obtain Lyb5-specific antiserum.

A new approach to obtain antiserum specific for Lyb5.2 allotype, was developed. Mice of DBA/2 inbred strain, bearing Lyb5.1 allotype, were made unresponsive to cellular antigens of Lyb5-CBA/N mice by specific tolerance induction. These tolerant recipients were further immunized with suspensions of splenocytes from CBA mice which contain Lyb5.2 + B lymphocytes. Thus obtained Lyb5.2-specific antiserum was cytotoxic for 25-30% of CBA or BALB/c (both Lyb5.2 + allotype) but not for CBA/N spleen cells. When splenocytes of BALB/c mice preimmunized with polyvinylpyrrolidone (T-independent type 2 antigen) were treated with this antiserum, 50% decrease in numbers of antibody-forming and antigen-induced non-specific immunoglobulin-forming cells was documented. We conclude that, during humoral immune response to T-independent type 2 antigen, not only antibody-forming cells but also non-specific immunoglobulin-forming cells are recruited from the Lyb5 + B cell pool.

[Isolation of human B-cells, specific for viral peptides, using magnetic spheres]. / Vydelenie B-kletok cheloveka, spetsifichnykh k virusnym peptidam, s pomoshch'iu magnitnykh sharikov.

A method for isolation of human B cells specific to viral peptides using magnetic beads has been developed. The method was used for the isolation from human tonsils of B cells specific to HIV-1 gp 120 MN-24 peptide (residues 302-322) and to Epstein-Barr virus 107 EBNA-1 peptide. The cells selected with beads were transformed by Epstein-Barr virus. The resultant cell cultures produce human immunoglobulin antibodies to HIV-1 MN-24 and to Epstein-Barr EBNA-1 p107 peptides.

[Immunosuppression induced by respiratory viruses (influenza virus, adenovirus) in mice]. / Ugnetenie respiratornymi virusami (gripp, adenovirus) immunogo otveta u myshei.

The influence of respiratory viruses (adenovirus, influenza virus) on humoral immune response to heterologous T-dependent and T-independent antigens was studied. It was shown that inoculation of mice by the influenza virus (A/PR8/34-A/PR/8) 3 days before sheep red blood cells administration led to the inhibition of antibody forming cell (AFC) and immunoglobulin, forming cell (IFC) increase on 69% and 59% respectively. Adenovirus type 6 induced the similar suppression of AFC and IFC formation. Thus, viruses induced immuno-suppression, which was polyclonal. It was also shown that virus of one strain (type) could inhibit immune response to another strain (type) of virus. The immune response to T-independent antigen was not suppressed. The virus-induced immunosuppression was dependent on: 1) the infectivity of respiratory viruses, 2) the route of virus and heterologous antigen injection, and 3) the interval between the viruses and antigen inoculation.

Role of antigen-binding B lymphocytes in the formation of antigen-dependent nonspecific immunoglobulin-forming cells.

A role for antigen-binding B cells in the induction of antigen-dependent B cells producing antigen-nonspecific immunoglobulins (nIFC) is analyzed. An in vitro immune response system was used; sheep red blood cells were employed as the antigen. It is shown that cocultivation of antigen-binding B cells with resting naive splenocytes in the presence of homologous antigen resulted not only in an increase in the number of antibody-forming cells (AFC), but also in a sharp increase in nIFC. Cocultivation of G0 splenocytes with primed B cells depleted for antigen-binding cells did not affect AFC induction, and only slightly augmented the number of nIFC. It is suggested that some of the nIFC appearing after antigenic stimulation arise from resting B lymphocytes in response (direct or indirect) to nonspecific B-cell factor(s).

[The hemodynamic support of physical loading in patients with mitral valve prolapse]. / Gemodinamicheskoe obespechenie fizicheskoi nagruzki u bol'nykh s prolabirovaniem mitral'nogo klapana.

Bicycle ergometry and echocardiographic studies were carried out in 29 patients aged 17-29 years with first revealed mitral valve prolapse (MVP) without any signs of mitral regurgitation. According to bicycle ergometry, the patients manifested changes in hemodynamics pointing to dysfunction of the cardiovascular system. Echocardiography discovered a reduction of the mass and a rise of the rate of contraction of the circular fibers of the left ventricle, evidence of the myocardial genesis of the hemodynamic changes. Comparison of the findings of bicycle ergometry and echocardiography allowed a conclusion about the necessity of the follow-up of patients with MVP in spite of the high level of threshold load and the lack of the clinical signs of heart failure.

[Monoclonal antibodies to ceruloplasmin and their use for immunoenzyme assay of this protein]. / Monoklonal'nye antitela k tseruloplazminu i ikh ispol'zovanie dlia immunofermentnogo analiza étogo belka.

The hybridoma technology was used to produce monoclonal antibodies to human ceruloplasmin. The antibodies were found to be related to Ig G1. Using these monoclonal antibodies for PAP-ELISA of ceruloplasmin, it was possible to determine 10(-11) M of the protein. Monoclonal antibodies coupled to CNBr-Sepharose were used for the rapid one-stage purification of ceruloplasmin from human placental serum. Ceruloplasmin obtained by this method contained no type 2 copper normally detected in protein preparations by conventional methods.

[The dynamics of Treponema-specific blood immunoglobulins in early forms of syphilis]. / Dinamika treponemospetsificheskoi immunoglobulinemii pri rannikh formakh sifilisa.

The study has involved 73 patients with syphilis, 31 female and 42 male ones, aged 18-42. Four of these suffered from primary seronegative, 14 from primary seropositive, 21 from secondary fresh, 22 from secondary recurrent, and 12 from early latent seropositive syphilis. Peripheral blood sera were under study. Treponema-specific antibodies of the IgM and IgG classes were titered by enzyme immunoassay. The detected changes in Treponema-specific immunoglobulinemia are in good correlation with clinical staged pattern of syphilis and antiinfectious immunity status.

Preparation of Hybridomas producing monoclonal antibodies against human interferon.

To prepare hybridomas secreting monoclonal antibodies (MoAb) against human alpha-interferon (alpha-IFN), BALB/c mice were immunized with IFN produced in Namalwa cells. Native alpha-IFN, as well as partially purified or on cellulose adsorbed alpha-IFN preparations were used for immunization. Seven hybridomas continuously secreting IgG against human alpha-IFN were prepared by fusion of splenocytes from immunized donors with the mouse myeloma cells. MoAb reacted in ELISA as well as in neutralization test with human lymphoblastoid, leukocytic and recombinant alpha-IFN.

[Humoral immune response to the influenza virus: the fractionation of sera from immune animals]. / Gumoral'nyi immunnyi otvet k virusu grippa: fraktsionirovanie syvorotok immunnykh zhivotnykh.

The studies demonstrated that antibody synthesis under conditions of the investigated fatal influenza infection differs in certain parameters from that in non-fatal infection. The mouse-pathogenic A/PR8/34 strain of influenza virus actively induced synthesis of antibodies detectable both by HI and NT. The less pathogenic A/Krasnodar/101/59 strain induced synthesis of antibodies detectable by NT sufficiently well but was a poor inducer of antibodies inhibiting virus hemagglutination of chick erythrocytes. Antibodies detectable by HI and NT appear to represent different molecules of immunoglobulins which differ in their immunochemical properties. These antibodies could be separated by means of affinity chromatography on an immunosorbent. The results of the above studies confirm that the protective and virus-neutralizing activity of an immune preparation in passive immunization is determined by the qualitative and quantitative composition of antibodies in a given preparation. The ratio of virus-induced antibodies may possibly determine the severity of the course and outcome of primary influenza infection.

[Role of G0- and G1-splenocytes and antigen-binding lymphocytes in the production of antigen-dependent nonspecific immunoglobulins]. / Rol' G0- i G1-splenotsitov i antigen-sviazyvaiushchikh limfotsitov, produtsiruiushchikh antigenzavisimye nespetsificheskie immunoglobuliny.

Immunization of irradiated and syngeneic splenocyte-treated CBA mice with bovine red blood cells stimulated the formation of both antigen-producing cells (APC) and antigen-dependent nonspecific immunoglobulin-producing cells (NIGPC). The injection of G0-cell-enriched, instead of normal, splenocytes (together with bovine RBC) to irradiated mice reduced by half the production of antigen-dependent NIGPC. Thus, it is evident, that some of them are formed from preexisting blast cells (G1). An additional removal of antigen-binding cells (ABC) from G0 lymphocyte population produced a still greater reduction in NIGPC formation (the increment was 92.3% lower than in the control). It is concluded that antigen-dependent NIGPC are formed both due to specific antigen-stimulation of G0 cells aid to nonspecific stimulation of blast cells with factors produced by antigen-stimulated T-cells.

[Relation of the formation of producers of antigen-dependent nonspecific immunoglobulins to the dose of T-dependent and T-independent antigens]. / Zavisimost' obrazovaniia produtsentov antigenzavisimykh nespetsificheskikh immunoglobulinov ot dozy T-zavisimogo i T-nezavisimykh antigenov.

The dependence of the production of antibody-forming cells (AFC) and non-specific immunoglobulin-forming cells (nIFC) on the doses of T-dependent (sheep red blood cells, SRBC) and T-independent (polyvinylpyrrolidone, PVP and pneumococcal polysaccharide SSS III) antigens was investigated. The immunization of BALB/c mice with immunogenic or subimmunogenic doses of SRBC and PVP induced a marked increase in the number of antigen-dependent nIFC. In contrast, the injection of any SSS III doses did not influence the amount of nIFC, although a specific immune response to SSS III was quite obvious. Thus, two T-independent antigens, type II, differ in their ability to induce non-specific immune reactions. The experiments on simultaneous administration of monoclonal anti-Thy-1.2 antibodies and PVP or SSS III to mice have demonstrated that these differences were not related to T-suppressor activity. The possible role of T helpers in the immune response to T-independent antigens is discussed.

[Formation of cells secreting antigen-dependent nonspecific immunoglobulins during immunization of mice with 2 T-independent antigens]. / Obrazovanie kletok, sekretiruiushcikh antigenzavisimye nespetsificheskie immunoglobuliny, pri immunizatsii myshei dvumia T-nezavisimymi antigenami.

Immunization of BALB/c and C3H/A mice with T-independent antigens (Vi-antigen of Salmonella typhi and polyvinyl pyrrolidone PVP350) induces the appearance of both antibody-forming cells (AFC) and a sharp increase in the number of cells forming nonspecific immunoglobulins (nIFC). This effect is not related to mitogenic properties of the antigens. The number of nIFC formed after simultaneous injection of both T-independent antigens does not differ from that of nIFC formed during immunization with each of these antigens alone. Thus, there was no summation of nIFC number after injection of two T-dependent or one T-dependent and one T-independent antigens. The mechanisms of nIFC formation under the influence of T-dependent and T-independent antigens are discussed.

[Role of antigen-binding cells in the formation of producers of antibodies and antigen-dependent nonspecific immunoglobulins]. / Rol' antigensviazyvaiushchikh kletok v obrazovanii produtsentov antitel i antigenzavisimykh nespetsificheskikh immunoglobulinov.

The role of antigen-binding cells in the formation of antigen-dependent nonspecific immunoglobulin producers in the Mishell-Dutton system was investigated. It was shown that the main part of antigen-dependent nonspecific immunoglobulin producers arise from the cells bearing antigen-specific receptors (as well as antibody producers). The nature of antigen-specific receptors of the precursors of antibody-forming and antigen-dependent nonspecific immunoglobulin-forming cells is discussed.