[Report on the fifth International AIDS Congress of June 4-9, 1989 in Montreal, Canada].
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Acquired Immunodeficiency Syndrome↗
Biomedical subjects
R Grunow
Publications and source records attributed to R Grunow.
Occurrence of hybridomas producing multispecific IgM in human x (human-mouse) fusions with lymphocytes from different human immune compartments.
Human lymphocytes derived from peripheral blood, the spleen and lymph nodes were fused to the HAT-sensitive heteromyeloma cell line CB-F7. The Ig-producing initial cell lines were selected, and the supernatants were further analyzed for specific antigen binding (ELISA). IgM-antibodies were found which reacted with self- and non-self antigens of different molecular origin (nucleotides, proteins, carbohydrate structures). These antibodies were called multireactive (multispecific). The multispecific IgM-producing human hybridomas occurred with higher frequencies in the spleen (6.9% of IgM-producers) cell fusions than in experiments where peripheral blood-derived lymphocytes were fused (2.7%). There were no hybridomas producing multireactive antibodies detected in fusion material from lymph nodes. The greatest number of multireactive IgM was seen when PBL from SLE or anti-HIV-positive patients were hybridized to CB-F7 cells. Representative cell lines were cloned and recloned. The multireactivity of the IgM produced by really monoclonal cells, however, was preserved.
Animals↗
[Monoclonal anti-HIV antibodies in HIV/AIDS research. Diagnosis and therapy].
It will be given an overview on several aspects of structure proteins of the HIV and monoclonal antibodies against these. Monoclonal antibodies (mabs) are helpful in research on structure of the virus and pathogenesis of AIDS. A lot of mabs from mice to env, gag and pol gene products has been established. Published human mabs were directed to gp41 and p25 structures of HIV. In future mabs could be used as helpful therapeutical agents especially as immunotoxins against infected cells or as vaccines in form of antiidiotypic mabs.
Acquired Immunodeficiency Syndrome↗
Development of specific human mab's by a small scale electrofusion technique: the influence of some physical and chemical factors on hybridoma yield of human peripheral blood lymphocytes XCB-F7 fusions.
A fusion chamber and an appropriate procedure are described which allow to fuse a sample of 15 to 25 microliters of cell suspension every two minutes. The cells can be observed throughout the process. They are not exposed to mechanical stress after the fusion pulse. Electrofusion between the heteromyeloma line CB-F7 and human mononuclear cells from peripheral blood of immunized donors is shown to provide stable hybridomas producing IgG against tetanustoxin. Pronase treatment, calmodulin, PEG, lanthanum and a number of variations in the fusion conditions were investigated as to whether they influence physical fusion of the cells, hybridoma yield, and immunoglobulin production.
Animals↗
Cryopreservation of newly formed human and mouse hybridoma cells.
The aim of this study was to develop an optimal technique for cryopreservation of newly formed human and mouse hybridoma cells immediately after fusion. It was shown that human hybridoma cells could be frozen most successfully at a cooling rate of 5 K/min whereas mouse hybridomas at 1 K/min. The percentage of FCS in cryopreservation media (30 or 90%) had no influence on the recovery of both types of hybridomas. For testing the efficiency of freezing, the fusion rate (number of growing and Ig producing hybridomas), the ratio of IgM:IgG-producing initial hybridoma lines and yields of specific wells were analysed. Comparable results were registered when optimally cryopreserved and non-frozen material was studied. Optimization of the cryopreservation of newly formed hybridomas may be of methodological importance, especially when a time-consuming screening for antigenic specificities must be carried out.
Animals↗
Establishment of human Ig producing heterohybridomas by fusion of mouse myeloma cells with human lymphocytes derived from peripheral blood, bone marrow, spleen, lymph node, and synovial fluid. Effect of polyclonal prestimulation and cryopreservation.
50 fusion experiments were carried out to analyse heterohybridization efficiencies on mouse myeloma cells of the P3 X63 Ag8/653 line with human lymphocytes derived from peripheral blood, bone marrow, lymph node, spleen or synovial fluid. We found higher yields of growing and human Ig-producing hybridoma lines when lymphocytes from spleen or lymph node were fused. Although primary hybridomas could be established from fusions with bone marrow-derived cells, only in nine out of 1616 initially seeded wells was Ig production registered. Four fusions using immune cells from synovial fluid were made without success. Independently of the source of lymphocytes pokeweed mitogen (PWM) prestimulation had no enhancing effect on the percentage of wells with cell growth and this did not alter the IgM:IgG ratio in primary hybridomas (9:1), although cells from all compartments used here (with the exception of bone marrow cells) could be stimulated with PWM to produce both IgG and IgM in cultures. Cryopreserved lymphocytes from different sources could be used for fusions with comparable results registered for the fresh material.
Animals↗
The high efficiency, human B cell immortalizing heteromyeloma CB-F7. Production of human monoclonal antibodies to human immunodeficiency virus.
This paper describes the construction of a new heteromyeloma cell line designated CB-F7. The cell line was derived from xenogeneic somatic cell hybridization between normal human B lymphocytes and the murine HAT-sensitive P3X63Ag8/653 cell line. CB-F7 cells were characterized by rapid cell growth (doubling time about 16 h) and high cloning efficiencies in culture medium supplemented with 10% or 5% fetal calf serum, respectively. The karyotype of the cells consists of about 75-78 chromosomes as well as two chromosomal fragments. Fusions of the cells with human peripheral blood cells resulted in approximately 2-6 clones per 10(5) seeded lymphocytes. Furthermore, the cells are ouabain resistant and therefore suitable for fusions with EBV-transformed lymphoblastoid cell lines. Using CB-F7 as the parental cell line a number of specific human mAb producing hybrids were established. For the first time, we describe here the generation of hybrids secreting human monoclonal antibodies to human immunodeficiency virus (HIV). Two monoclonal antibodies of IgG type and one of IgM type reacted with the major core protein p25 and one IgG antibody reacted with the transmembrane protein gp41.
Antibodies, Monoclonal↗
A rapid and sensitive enzyme immunoassay for Cu/Zn superoxide dismutase with polyclonal and monoclonal antibodies.
An enzyme immunoassay for the quantification of human Cu/Zn SOD in serum, urine and erythrocytes was developed applying monoclonal and polyclonal antibodies. The one-step assay is completed within 30 min and enables the detection of 0.3 microgram Cu/Zn SOD per litre. A Cu/Zn SOD concentration of 46 +/- 21.5 micrograms/l and of 1 +/- 0.6 micrograms/mmol creatinine was determined in the serum and the urine, respectively, of healthy individuals. A content of 15 +/- 1.7 ng Cu/Zn SOD was found in 10(6) erythrocytes. Patients with Down's syndrome exhibited a 3.8-fold, a 2-fold and a 1.6-fold higher concentration of Cu/Zn SOD in their serum, urine and erythrocytes.
Antibodies↗
A myelopeptide from unstimulated bone marrow cells with immunomodulatory activity in lymphocyte cultures from healthy donors and patients with hypogammaglobulinemia and active lupus erythematosus.
A myelopeptide (SAP) was derived from culture supernatants of unstimulated animal bone marrow. SAP consists of a group of peptides with a molecular weight of about 2000 D, having a broad variety of biological activities. Testing immunoregulatory properties of the purified factor Petrov, Mickhailova & Zacharova [(1971). Immunoglobin synthesis in syngenic cells of different lymphoid tissues. J. Immun., 106 1086-1089] found enhanced antibody production in mice (SAP-stimulator of antibody production). We show here that the substance could induce expression of activation markers on human lymphocytes (4F2, HLA-class II antigens, thermostable SE rosette formation) and potentiate their appearance in combination with mitogens (PWM, PHA, Con A). Although SAP was not mitogenic for itself, it enhanced lectin-induced 3H-thymidine incorporation and T-cell-dependent B-cell differentiation in a dose-dependent manner. The factor was able to reconstitute disturbed PWM-driven Ig synthesis in lymphocyte cultures derived from two patients with hypogammaglobulinemia and a healthy non-responder to PWM. On the other side, SAP potentiated the inhibitory activity of PWM on elevated spontaneous IgG secretion in cultures derived from patients with active SLE. Findings of this study indicate immunomodulatory capacity of SAP on human peripheral blood lymphocytes possible via T-cell activation. The results suggest a potential therapeutic application of SAP in patients with disturbances in the T-dependent B-cell differentiation.
Agammaglobulinemia↗
Feeder cells from different sources and conditioned media for recloning of human--mouse and mouse--mouse hybridomas.
The use of different feeder cell layers (peritoneal macrophages from mice and rats, spleen cells and thymocytes from mice) for recloning of human--mouse and mouse--mouse hybridomas has been described. Optimal numbers of feeder cells from different sources required for high cloning efficiencies were determined. It was possible to use cryopreserved rat and mouse macrophages as feeders for cell cloning. However, the resulting cloning efficiency was much lower in comparison to the fresh material. Culture supernatants from human endothelial cells (added in a final concentration of 40% v/v) and from chicken embryo fibroblasts (25%) could replace the feeder cell layer in recloning experiments with both human--mouse and mouse--mouse hybridomas. Therefore, conditioned media (prepared in large quantities) may be used for generating standardized conditions for high-efficient cloning and recloning of hybridoma cell lines.
Animals↗
[Modification of in vitro immune mechanisms by various glucocorticosteroid preparations--studies of lymphocytes in healthy probands and autoimmune patients].
The influence of steroids on lymphocyte responses was tested in vitro. The inhibition of early events of lymphocyte stimulation and cell proliferation after PHA stimulation was estimated. We found, however, different individual responses of immune cells of various donors to the preparations dexamethasone, prednisolone, hydrocortisone or methylprednisolone in both in vitro models tested. Steroid treatment of lymphocyte cultures resulted in both inhibition and enhancement of PWM-driven Ig synthesis in cultures derived from different test persons. In experiments with lymphocytes from patients with acute systemic lupus erythematosus we found a dramatically enhanced PWM-stimulated IgG synthesis which is disturbed in cultures of these patients. The individuality of steroid sensitivity of lymphocyte cultures of healthy test persons and the abnormal reactivity of SLE lymphocytes were discussed in relation to therapeutical applications.
Adult↗
[Pan T, T4 and T8-positive lymphocytes in the peripheral blood of patients with systemic lupus erythematosus].
In a group of 23 patients with systemic lupus erythematosus by means of monoclonal antibodies differentiation markers were investigated on peripheral blood T-lymphocytes. Individual specific differences in the percentage of T1-, T3-, T4- and T8-positive cells were found, indicating various possibilities of immunological disorders in this disease. Furthermore, in comparison with healthy donors, an increased number of T4-, T8-double stained cells was detected in the peripheral blood of patients with systemic lupus erythematosus. Normally, this cell phenotype can be detected in the thymus only. Therefore, it becomes necessary to perform parallel and simultaneous determinations, respectively, of several T cell markers in order to gain a more exact insight into the disturbances of the immune system and to exclude incorrect interpretations of the phenotypical distribution of T cell markers.
Fluorescent Antibody Technique↗
Cell biology of human IgM-producing hybridomas derived from a fusion of human spleen lymphocytes with mouse myeloma cells.
Hybrids were derived from the fusion of mouse myeloma cells with human spleen cells from a patient with active idiopathic thrombocytopenia. Of 288 initially seeded cultures, 186 were found to produce human Ig. The growth and Ig production rates, cloning efficiencies using different feeder layers and the karyotype were determined for 9 clones that stably produced human monoclonal IgM (2-100 micrograms/ml) for at least 9 months. All cells of the Ig-producing hybridoma clones were positive for cytoplasmic-Ig, whereas only 20-65% of cells expressed surface Ig (mu and chains). Human monoclonal antibodies in mass cultures were derived in serum-free PRMI 1640 medium. Two clones produced human IgM (nearly 2 mg/ml) in the ascitic fluid of nude mice. Feeder cells of peritoneal macrophages from Balb/c mice enabled more efficient recloning of human x mouse hybrids than did thymocytes. Nearly all subclones derived from 2 clones were found to produce the same monoclonal antibodies as the parental lines. Information on the individual parameters of a hybridoma cell line may be helpful in the large-scale production of human monoclonal antibodies.
Animals↗
Functional properties of lectin-induced thermostable SE rosette (TSER)-forming cells derived from human peripheral blood.
Mitogens are able to stimulate T cells in human peripheral blood lymphocyte cultures to form thermostable rosettes with sheep erythrocytes (TSER). Previously we characterized TSER-forming cells as BL/T2-, DR- and 4F2-positive, e.g. activated T lymphocytes. In this study the evidence is presented that mitogen-induced TSER-forming cells are the responding, proliferating lymphocytes. We studied the sequential activation of human lymphocytes by various mitogens. PHA-induced TSER-forming cells could not been further activated neither by PWM nor by Con A. DNA synthesis rate in Con A-preactivated lymphocytes was enhanced by PHA but not by PWM. PWM-induced TSER-forming cells, however, were further activated as well by PHA as by Con A. These data suggest the heterogeneity of the pathways by which different mitogens activate human immune cells. PWM- and PHA-induced TSER-forming cells could support B lymphocyte differentiation, when PWM was added to the cultures, whereas Con A-preactivated cells failed, indicating different functional activities induced by mitogenic lectins.
Concanavalin A↗
[In vitro immunization for the production of antibodies to tetanus toxin and toxoid. 2. Antibody production in lymphocyte cultures].
The aim of this work was to optimize the antigen-specific prestimulation of human lymphocytes for further hybridization to produce human monoclonal antibodies. In vitro stimulation of human peripheral blood lymphocytes with purified Tetanus Toxoid (TT) resulted both in an increased amount of secreted specific antibodies and in an enhancement of the portion of anti-TT antibodies in the level of total immunoglobulin secretion. Both IgG and IgM antibodies were found. Donors who had not been boostered in vivo with TT for more than 10 years also showed an antibody response to the antigen in vitro. Five days after in vivo boostering we found in lymphocyte cultures derived from 3 donors an enhanced antibody synthesis in the response to TT and also an increased portion of specific antibodies in the spontaneously secreted immunoglobulin in unstimulated control cultures.
Antibodies, Bacterial↗
In vitro immunization for antibody production against tetanus toxin and toxoid. 3. Recombinant gamma interferon and indomethacin support specific antibody production induced by antigen-pulsed immobilized monocytes.
Specific antibody production against Tetanus Toxin by in vitro cultured human peripheral blood lymphocytes may be enhanced, when monocytes were immobilized onto FCS-coated culture plates and then pulsed with Tetanus Toxoid. Using this model several immunomodulators were tested for their influence on in vitro immunization efficiency. A combination of recombinant gamma interferon and indomethacin added during antigen presentation to lymphocytes, was able to potentiate specific antibody response.
Antibody Formation↗
Influence of hydroxyurea on lectin-induced early and late events in peripheral blood lymphocyte cultures.
Activation events induced by lectins in human lymphocyte cultures differed not only in time kinetics of their appearance but were also in a different manner inhibited by hydroxyurea (HU). 4F2 and Tac expression, thermostable sheep erythrocyte rosette formation, cell volume distribution changes and the enhancement of the purine metabolic rate [( 3H]adenine incorporation) induced by PHA, Con A or PWM were not influenced by HU treatment, suggesting G1-phase dependency of these markers. The decrease in the mitogen-induced marker expression after 48 h of incubation with HU can be explained by the unability of activated cells to process the cell cycle, to divide and to become newly positive cells. Mitogen-induced RNA synthesis was partially, DNA synthesis, PWM-induced Ig synthesis and lectin-mediated HLA class II antigen expression were totally inhibited by HU. It holds especially for the DR antigen that its appearance in a polyclonal model of lymphocyte activation occurs in a later (postmitotic G1) phase of the cell cycle. The inhibitory effect of HU on late activation parameters could be removed after washing the cells. Mitogen-activated, HU-treated PBL cultures after removing HU continued cell cycle progression without requirement for further addition of lectin.
Adenine↗
[Masking of pan-T-cell markers in patients with autoimmune diseases].
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Autoantibodies↗