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Biomedical subjects

S Tamir

Publications and source records attributed to S Tamir.

34 records · Page 2Linked to original sources
PubMed1988

The presence of non-isotype-specific antibodies in polyclonal anti-IgE reagents: demonstration of their binding to specifically selected Epstein-Barr virus-transformed cell lines.

Polyclonal anti-human IgE reagents were earlier shown to contain variable amounts of nonisotype-specific antibodies depending on the strategy used for their preparation. The presence of these antibodies in two commercial anti-IgE reagents was demonstrated in this work by (a) their binding to human Ig-surface-positive lymphoblastoid cells specifically selected by one of the polyclonal anti-human IgE reagents and (b) their binding to the non-IgE immunoglobulins secreted by those lymphoblastoid cells. Peripheral blood B lymphocytes from two normal and two atopic patients were immortalized with Epstein-Barr virus (EBV) and then selected for cells that rosette with anti-IgE-coated erythrocytes. Selection was repeated four times and cells were then cloned. The cloned cells formed rosettes and their supernatants agglutinated erythrocytes coated with rabbit anti-IgE. The immunoglobulins of these clones were positive in an ELISA for IgE, using two different polyclonal anti-human IgE reagents. They were shown, however, to be 19 S IgMs. This discrepancy was due apparently to substantial contamination of anti-non-IgE-isotype-specific antibodies in the polyclonal anti-IgE reagents used both in the selection of cells and in the ELISA. The human monoclonal B-cell lines which were applied here as targets amplified the non-IgE-isotype specific antibody contamination present in the polyclonal anti-human IgE reagents. Because of the normally very low frequency of IgE-positive cells, the use of polyclonal anti-IgE reagents to detect these cells has to be carefully evaluated.

Animals↗
PubMed1986

A protective human monoclonal IgA antibody produced in vitro: anti-pneumococcal antibody engendered by Epstein-Barr virus-immortalized cell line.

Human lymphocytes that produce anti-pneumococcal antibodies were separated and immortalized by Epstein-Barr virus and then cloned. One clone (NAD-Sel) produces an IgA, kappa antibody which is specific for the polysaccharides of type 8 pneumococcus, while not reactive with any of the polysaccharides derived from 24 other pneumococcal strains. The antibody, which is present in the cell supernatant as monomer and polymer, binds to protein A and does not fix complement. When incubated in vitro with type 8 pneumococci, it induces direct killing and increases the opsonization of these bacteria by mouse macrophages.

Animals↗
PubMed1985

The coating of erythrocytes with detergent-solubilized molecules: a general method for improved coupling of antigens and antibodies.

A method is described by which different antigens and antibodies in solutions containing deoxycholate (DOC) may be coated to glutaraldehyde (GA)-fixed erythrocytes. This method, based on the use of CrCl3, yields erythrocytes which preserve the antigenicity and antibody activity of the coated molecules and can thus be applied to various assays in which indicator red cells are required. The sensitivity of the assays increases when these red cells are used. The cells form rosettes with the appropriate receptor positive cells. They sediment in Ficoll-Isopaque as do native erythrocytes and do not aggregate spontaneously even when coated at very high or low concentrations of CrCl3 or antigen. This coupling method, which is performed in the presence of DOC, and which requires only a small amount of antigen, is proposed for the coupling of cell membrane dissolved antigens.

ABO Blood-Group System↗
PubMed1985

Human monoclonal antibodies produced by Epstein-Barr virus transformed cell lines bind protein A.

Epstein-Barr virus (EBV) is a polyclonal T-independent activator of viral receptor positive human B lymphocytes. Lymphocytes infected in vitro with the virus are transformed into immortalized cell lines [Nilsson, K, and Klein, G. (1982) Adv. Cancer Res. 37, 319]. In this way human cell lines that secrete specific IgM, IgG and IgA monoclonal antibodies are established. Protein A is also a polyclonal T-independent B cell activator [Langone, J. J. (1982) Adv. Immunol. 32, 157], the targets of which are surface immunoglobulin and C3d receptor positive cells, as are the targets of EBV. We found that almost all (16 out of 17) of the specific monoclonal antibodies (IgM, IgG and IgA) produced in vitro by EBV cell lines bind protein A. Unlike these in vitro produced antibodies, a substantial fraction of the immunoglobulins in human serum does not bind protein A. Thus, those plasma cells which in vivo secrete protein A nonbinding immunoglobulins originate from precursors of B cell that were EBV noninfective. Alternatively, during in vivo B differentiation some immunoglobulins undergo a change from protein A binding to protein A nonbinding molecules.

Adult↗
PubMed1985

Continuous in vitro production of IgA by a human colostral immortalized cell line.

Breast milk samples from 8 postpartum mothers were collected and incubated with Epstein-Barr virus, immortalizing their B-lymphocytes and giving rise to lymphoblastoid cell lines. Three samples showed no growing lymphocytes and five samples gave rise to transformed cell lines. IgA positive cells were selected from one such cell line by rosetting with anti-IgA-coated sheep erythrocytes. The emerging cell line was similarly reselected giving rise to an IgA-surface positive lymphoblastoid cell line. Monoclonal IgA-producing cell lines were obtained by cloning in the soft agar method. IgA is continuously secreted by the cell lines which have been growing in vitro for more than 24 mth.

Antibody-Producing Cells↗
PubMed1985

Solubilization of cell-membrane-antigen and its coupling to fixed and non-fixed erythrocytes.

Human type-A erythrocytes were lysed and solubilized with 0.3% sodium deoxy cholate (DOC). The solubilized membranes were coated onto human type-O glutaraldehyde fixed erythrocytes. The coated erythrocytes were found to agglutinate specifically by anti-A antibodies. Furthermore, solubilized membranes could be successfully coated to non-fixed erythrocytes after dialysing out the DOC. Quantitative absorption assays showed that the density of type-A blood group antigen on the coated erythrocytes was higher than the density of this antigen on human type-A erythrocytes. Erythrocytes that are coated with cell membrane antigens can be used to assess specific antibody titres and also can be applied for a sensitive selection and separation of rosetting lymphocytes that secrete specific antibodies against these antigens.

ABO Blood-Group System↗
PubMed1984

Human anti-pneumococci antibody produced by an Epstein Barr virus (EBV)-immortalized cell line.

Anti-pneumococcal antibody-producing lymphocytes from a patient who was immunized with Pneumovax (a polysaccharide mixture from 14 different pneumococci types) were selected and immortalized with Epstein Barr virus (EBV). The cells of the emerging cloned line were shown on a single cell level assay to secrete an IgM, kappa anti-pneumococci antibody. This human monoclonal antibody bound specifically to polysaccharides of pneumococci type 8, but did not bind to any of the other 13 pneumococci types that were present in the Pneumovax mixture. In competition experiments, inhibition of binding occurred with polysaccharide type 8 and Pneumovax only. This diploid cell line has actively secreted the specific antibody (2.8 micrograms/10(6) cells/ml) now for more than 9 mo. In patients, increased anti-pneumococcal antibody titers generated by active immunization with Pneumovax improves resistance to the corresponding bacterial infection. Production in vitro of human monoclonal antibodies directed toward bacterial and viral antigens enables a new possible approach for passive immune protection.

Antibodies, Bacterial↗
PubMed1983

A sensitive assay to evaluate antistreptococcal antibodies using an in vitro-produced monoclonal human rheumatoid factor.

A human monoclonal rheumatoid factor (RF) produced in vitro by an Epstein-Barr virus (EBV) immortalized cell line is used in an assay to detect and quantitate antibodies directed against streptococcal antigens in human sera. The assay, based on the binding of the rheumatoid factor to IgG-immune complexes, applies this monoclonal antibody in its iodinated form. The assay can easily quantitate IgG-antistreptococcal antibodies and enables the quantitation of these specific antibodies in sera that contain even very low titers.

Antibodies, Bacterial↗
PubMed1982

A sensitive method for detecting cells coated with antibodies with human monoclonal rheumatoid factor produced in vitro.

A human monoclonal rheumatoid factor (RF) produced in vitro by an Epstein-Barr virus immortalized cell line has been used to detect and quantitate antibodies specifically bound to cells. The RF is purified from cell culture supernatant by a simple procedure and then radiolabeled. In this assay, the binding of 125I-labeled RF to cells coated with specific antibodies is determined. This RF method detects very low titers of antibodies directed against specific cellular antigens and also minorities of antibody coated tumor cells in mixtures with identical non-coated cells. The advantages of this unique human monoclonal antibody are discussed.

Animals↗
PubMed1982

Human monoclonal autoimmune antibody produced in vitro: rheumatoid factor generated by Epstein-Barr virus-transformed cell line.

The infection of selected lymphocytes from a rheumatoid arthritis patient with Epstein-Barr virus resulted in an immortalized cell line that secretes a monoclonal rheumatoid factor (RF). The cloned line has been growing for more than 24 months, and constantly produces a monoclonal IgM, lambda, 19S, RF (1-2 micrograms/ml/106 cells). The RF agglutinates human and rabbit IgG (but not IgM) and also protein A-coated erythrocytes, but fails to do so to mouse, goat and swine IgG-coated erythrocytes. When bound to immune complexes, this monoclonal RF dose not bind complement. In the cell supernatant RF is the only immunoglobulin and it comprises approximately 5% of the total proteins. The affinity of RF to aggregated human IgG, as detected in inhibition experiments, is higher than that of Fc receptors found on human non-T lymphocytes, K562 and Daudi cell lines.

Antibodies, Monoclonal↗
PubMedDate not supplied

Paradoxical interactions among estrogen receptors, estrogens and SERMS: mutual annihilation and synergy.

The phenomenon of mutual annihilation of action between 17beta estradiol (E(2)) and a selective estrogen receptor modulator (SERM), previously described in prepubertal rat diaphysis, epiphysis and uterus, has been investigated in ROS 17/2.8 rat osteoblastic cells and in transiently co-transfected cells in culture. In ROS 17/2.8 cells, the estrogen-induced marker enzyme creatine kinase B (CKB) was stimulated by raloxifene, tamoxifen and tamoxifen methiodide to a specific activity equal to or greater than that induced by 10 nM E(2). However, when a fully inhibitory dose of any of these SERMS was given simultaneously with E(2), no stimulation of CK activity resulted. Therefore, SERMS can be full agonists when acting alone, but complete antagonists to a super-physiological dose of estrogen. It is expected that excess tamoxifen would prevent the action of a SERM, but that the agonist activity of a SERM is abolished by 1000-fold less estrogen is a phenomenon without obvious explanation by classical pharmacology of competitive inhibition. To probe the mechanism of this interaction further, a ckb-CAT reporter plasmid, plus the human receptor expression plasmid, HEO, was transfected transiently into several cell types. In MCF-7 cells, a 1:10 ratio of E(2) to tamoxifen produced mutual annihilation, but the same ratio in ROS 17/2.8 or HeLa cells led to synergistic stimulation. In HeLa cells, co-transfected with the more efficient wild-type estrogen receptor plasmid, HEGO, synergy was demonstrated only at sub-saturation levels of HEGO. We speculate that, in the presence of estradiol and a SERM, not only active homodimers would be formed, but also hetero-dimers of estrogen-liganded and tamoxifen-liganded receptor monomers, depending on the molar ratio of their ligands and their relative affinities. The resulting hetero-dimer conformation would change the specific receptor surface for interactions with the growing number of co-activators and co-repressors, structural changes which could help to explain the mutual annihilation and synergy phenomena and their cell selectivity.

Antineoplastic Agents, Hormonal↗
PubMedDate not supplied

The influence of delivery rate on the chemistry and biological effects of nitric oxide.

Nitric oxide can be introduced slowly and steadily into aqueous solutions, including cell culture media, over extended periods of time via semipermeable Silastic (a registered trademark of the Dow Corning Corp.) polymer membranes. The rates of introduction are predictable and reproducible and can approach rates of nitric oxide production by stimulated cells, such as macrophages, that express inducible nitric oxide synthases. DNA damage in Chinese hamster ovary cells by membrane-delivered nitric oxide is comparable to that observed in the DNA of stimulated macrophages. Toxicity and mutagenicity of nitric oxide toward Salmonella typhimurium, toxicity of nitric oxide toward Chinese hamster ovary cells, and nitrosation of dimethylmorpholine are all more efficient when nitric oxide is delivered by membrane than when an equivalent amount of gaseous nitric oxide is added by syringe.

Animals↗
PubMedDate not supplied

Nitric oxide induces oxidative damage in addition to deamination in macrophage DNA.

Inflammatory cells such as phagocytes, neutrophils, and macrophages have been implicated in the pathogenesis of several forms of clinical and experimental tumor development. It is hypothesized that this process is mediated by the production of reactive species including NO., O2.-, H2O2, and ONOO- which inflict DNA damage. In this study, the role of NO. in combination with oxygen radicals in DNA damage was investigated. DNA deamination (xanthine) and oxidation [5-(hydroxymethyl)uracil (5HMU), 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FAPY-G), and 8-oxoguanine (8oxoG)] products were identified in the DNA of macrophages (RAW264.7) activated with Escherichia coli lipopolysaccharide (LPS) and mouse gamma-interferon (INF-gamma). The formation of these products was inhibited by N-methyl-L-arginine (NMA), a nitric oxide synthase inhibitor. NMA inhibited only the production of nitric oxide and had no effect on superoxide production. These results demonstrate that NO. plays a dual role in damaging the DNA of activated macrophages. Autoxidation of NO. leads to nitrosating species which cause deamination of bases. Reaction of NO. with O2.- leads to DNA oxidative damage due to the formation of peroxynitrite which may have HO.-like oxidizing potential. Another possible mechanism of oxidative damage by NO. could be the mobilization of free iron by NO. which could ultimately cause Fenton-type reactions. Therefore, nitric oxide not only leads to deamination of DNA bases but is also an obligatory factor in oxidative damage to DNA.

Animals↗