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P L Simon

Publications and source records attributed to P L Simon.

33 records · Page 2Linked to original sources
PubMed1986

Evidence for shared antigenic determinants on rabbit and human interleukin 1 (IL 1).

An antiserum to human interleukin 1 (IL 1) was prepared by immunizing a goat with the isoelectric point (pI) 6.9 type of IL 1 in Freund's complete adjuvant. Serum-mediated inhibition of the biological activity of IL 1 appeared within 4 wk after the first immunization, and showed a progressive rise in titer over a 9-mo period. The inhibitory moiety was purified by sequential ammonium sulfate fractionation and DEAE-Sephacel ion exchange chromatography, and the activity was found to co-purify with the IgG fraction of the serum. The antibody neutralized the biological activity of the pI 6.9 type of human IL 1 derived from either human placental tissue or human peripheral blood adherent cells, but did not neutralize the pI 5.2 type of IL 1 derived from either source. When used as an affinity reagent, the antibody selectively absorbed the pI 6.9 human IL 1, but not the pI 5.2 human IL 1. Furthermore, the antibody neutralized the pI 7.4 type of IL 1 derived from rabbit alveolar macrophages, but had no activity against the pI 4.6 IL 1 derived from the same source. No inhibitory activity against rat spleen cell-derived IL 1 or murine P388D1 cell line-derived IL 1 was detected. These experiments support the concept that the differing pI types of IL 1 derived from the same species are both biochemically and antigenically distinct molecules, and IL 1 of similar pI type derived from different species may share antigenic determinants.

Animals↗
PubMed1986

Serological characterization of acidic (pI 5.5) and neutral (pI 7.1) forms of 17kD and 35kD interleukin-1.

Human interleukin-1 (IL-1) was partially purified into the acidic and neutral 17,000 dalton forms and the acidic and neutral 35,000 dalton forms. Heterologous antiserum raised against purified human IL-1 (neutral, 17,000 daltons) neutralized only the neutral 35,000 dalton and 17,000 dalton isoelectric forms. Conversely, an antiserum raised against purified porcine acidic IL-1 (21,000 daltons) neutralized only the acidic 35,000 and 17,000 dalton forms. These results confirm earlier findings that the acidic and neutral species of IL-1 are not derived from the same gene. In addition, the results suggest that each isoelectric form of the 17,000 dalton IL-1 is probably derived directly from the corresponding 35,000 dalton IL-1.

Antigen-Antibody Complex↗
PubMed1986

The effects of interleukins and other soluble factors on T-lymphocyte colony formation.

When plated in semi-solid media, PHA-stimulated human peripheral blood mononuclear cells (PBMC) form discrete T-cell colonies. By contrast, Sephadex G-10 non-adherent (NA) cells (greater than 96% T lymphocytes) show virtually no clonal growth unless cocultured with soluble factors derived from either normal adherent cells or tumour cell lines. Purified IL-1 was able to initiate colony growth of mitogen-stimulated NA cells; cultures containing 20 U of human IL-1 yielded colony counts that were only slightly less than those with PBMC. In addition, recombinant IL-2, free of measurable IL-1, was able to provide the initiating signal required for clonal expansion. Both recombinant and lymphocyte-derived IL-2 were able to enhance the clonal growth of PBMC. Colony growth could be initiated by supernatants derived from short-term cultures of either monocytic (U937, HL60) or B-cell (Raji, Daudi) tumour cell lines. The abilities of these tumour cell lines to promote clonal responses did not correlate with their contents of either IL-1 or IL-2. By contrast, supernatants derived from either K562 (an erythroleukaemic line) or MOLT 4 (a T-cell lymphoma) cells did not provide the initiating signal.

Antibodies, Monoclonal↗
PubMed1986

Comparisons of 12-tetradecanoylphorbol-13-acetate (TPA) and PHA as mitogens in the T-lymphocyte colony assay.

12-O-tetradecanoylphorbol-13-acetate (TPA) has multiple effects on the capacity of human T-lymphocytes to form colonies in soft agar. This compound is directly mitogenic for T-lymphocytes; the optimal concentration (100 ng/ml) stimulates an average of 2862 +/- 583 colonies/7.5 X 10(5) cells plated. Furthermore, TPA can act synergistically with PHA to induce a greater number of colonies than can either mitogen alone. Sephadex G10 nonadherent (NA) cells can be directly stimulated by TPA; by contrast, these isolated T cells do not respond to PHA alone. These data indicate that the phorbol ester is able to provide an inductive signal for T cells, allowing them to respond to the plant lectin. Using T-cell subsets isolated by monoclonal antibodies and complement cytotoxicity, PHA is able to induce colony growth of T4 cells; T8 cells fail to respond unless cocultured with exogenous IL-2. TPA can directly stimulate colony formation by both subsets. In cultures stimulated with either TPA or PHA, approximately equal numbers of colonies are generated in the presence of IL-2, suggesting that T4 and T8 cells have similar proliferative capabilities. Phenotypic studies of cells contained in colonies showed differences between the two mitogens. With PHA, more than 98% are both T11 and T3 positive; by contrast, approximately one-third of the cells stimulated by TPA are T11 +, T3-.

Cell Separation↗
PubMed1985

A modified assay for interleukin-1 (IL-1).

A simple and reliable biological assay for interleukin-1 (IL-1) was developed, based on the production of interleukin-2 (IL-2) from the EL-4 murine T-cell lymphoma cell line, in the presence of 2-5 X 10(-7) M calcium ionophore A23187. The assay was generally performed in 2 stages ((a) IL-1-dependent IL-2 production, and (b) IL-2 assay) and took 36-48 h to complete. This assay was found to be 10-25 times more sensitive than the mouse thymus cell assay, was not sensitive to the presence of bacterial endotoxin, and had the advantage of not requiring the use of animal tissue as a source of cells. The assay was used in our laboratory to detect human, mouse, rat, and rabbit IL-1 of all isoelectric-point types.

Animals↗
PubMed1984

Calcium mediates one of the signals required for interleukin 1 and 2 production by murine cell lines.

The murine T lymphoma cell line EL-4 can be induced to produce interleukin 2 (IL-2) by concurrent stimulation with interleukin 1 (IL-1) and a T-cell lectin such as phytohemagglutinin (PHA) or concanavalin A (Con A). The results presented here demonstrate that the requirement for the lectin, but not IL-1, could be completely replaced by the calcium ionophore A23187. The optimal effective concentration of A23187 was found to be 2.5 X 10(-7) M, and the costimulating effect of IL-1 was dose-dependent. The stimulatory effect of A23187 was completely eliminated by incorporation of 5 mM ethylene glycol bis (beta-aminoethyl ether) N,N,N',N'-tetracetic acid (EGTA) in the culture medium, and this inhibition could in turn be reversed by addition of 5 mM CaCl2 to the medium. Release of IL-2 from IL-1/A23187-stimulated EL-4 was detected within 5 hr after initiation of the cultures, and both signals were required at the same time to initiate synthesis or release of IL-2. In addition, the calcium ionophore also augmented release of IL-1 from the P388D1 murine macrophage cell line. These results suggest that a calcium-mediated event may serve as a common mechanism for the induction of secretion of lymphokines and monokines from murine cell lines.

Animals↗
PubMed1981

The role of subcellular factors in pulmonary immune function: physicochemical characterization of two distinct species of lymphocyte-activating factor produced by rabbit alveolar macrophages.

T cell stimulatory factors produced by rabbit alveolar macrophages were investigated. Physicochemical characterization revealed that alveolar macrophages (harvested by bronchopulmonary lavage and stimulated in tissue culture with bacterial lipopolysaccharide) released 2 predominant species of lymphocyte-activating factor (LAF) with isoelectric points of 4.6 and 7.4, and m.w. of 14,400 and 11,600 daltons, respectively, as calculated by the Svedberg equation. Using C3H/HeJ mouse thymocytes (and in some instances nylon wool-purified nonadherent rabbit spleen or lymph node cells) as target cells, rabbit LAF was found to induce proliferative responses directly, as well as enhance proliferative responses to phytomitogens. Both LAF species were inactivated by heating, treatment with trypsin, or at low (2.3) pH. The pI 7.4 LAF was also unstable at high pH (9.0). The thymocyte stimulatory activity of both LAF species was not inhibited by the anti-proteases alpha-1-anti-trypsin, Traysylol (aprotinin), leupeptin, or pepstatin.

Animals↗
PubMed1980

Endogenous pyrogens made by rabbit peritoneal exudate cells are identical with lymphocyte-activating factors made by rabbit alveolar macrophages.

Purified endogenous pyrogen (EP), isolated in one laboratory from stimulated rabbit peritoneal exudate cells (PEC), was compared with purified lymphocyte activating factor (LAF), isolated in another laboratory from stimulated rabbit alveolar macrophages (AM). Both EP and LAF occurred in two forms, one with an isoelectric point (pI) of 7.3 to 7.4, the other with a pI of 4.6 to 4.7. Both forms of EP and LAF had m.w. of 13,000 to 16,000 daltons as judged by gel filtration. Both forms of EP had LAF activity, and both forms of LAF were pyrogenic. The pI 7.3 EP, which is known to have an -SH group essential for its biologic activity, bound to Thiol Sepharose columns and could be eluted with mercaptoethanol. The pI 7.3 LAF behaved in exactly the same way. Furthermore, antisera from three different goats were available that completely blocked the pyrogenicity of pI 7.3 EP in vivo; these sera also blocked the pI 7.3 LAF activity in vitro. The pI 4.6 EP and LAF did not bind to Thiol Sepharose columns, nor were they inhibited by any of the antisera that blocked the pI 7.3 EP and LAF. Moreover, isoelectric focusing of pI 4.6 EP in very shallow gradients revealed microheterogeneity with sharp peaks of EP activity observed at pH 4.6 and 4.7. Analysis for LAF activity showed an identical microheterogeneity. These results are consistent with the idea that EP and LAF are identical.

Animals↗
PubMed1980

Thymoma production of T cell growth factor (Interleukin 2).

Phorbol-12-myristate-13-acetate stimulates a subline of mouse EL-4 thymoma cells to produce, in vitro, in very high titer, T cell growth factor (Interleukin 2, IL 2). The EL-4-derived IL 2 has the same m.w. (30,000) and isoelectric point heterogeneity (pI 3.8-4.4) as the IL 2 produced by Con A-stimulated spleen cells. In addition, the thymoma-derived IL 2 exhibits the same spectrum of biologic activities as has been reported for spleen cell-derived IL 2.

Animals↗
PubMed1979

Biochemical relationship between murine immune interferon and a killer cell helper factor.

Concanavalin A and alloantigen-stimulated mouse spleen cells release into the supernatant at least two distinct antigen-nonspecific factors that enhance the generation of cytotoxic cells in vitro. As previously reported, analysis of the supernatant by ammonium sulfate fractionation, gel filtration, hydroxylapatite chromatography, and hydrophobic chromatographic showed one of the two killer cell helper factors (KHF) to be associated with thymocyte mitogenic factor. This report demonstrates the second KHF to be separable from thymocyte mitogenic factor but inseparable from type II (immune) interferon. In addition, this KHF exhibits the same sensitivity to exposure to pH-2 buffer as does immune interferon. The KHF activity of an unfractionated supernatant, which is greater than of either the TMF-associated or interferon-associated KHF alone, is the result of the additive activities of the two independently acting helper factors.

Ammonium Sulfate↗
PubMed1978

Biochemical relationship of thymocyte mitogenic factor and factors enhancing humoral and cell-mediated immune responses.

Supernatants from concanavalin A-induced mouse spleen cells contain soluble factors which: 1) augment the anti-sheep erythrocyte hemolytic plaque-forming cell response of nude mouse spleen cells, 2) facilitate the generation of antigen-specific cytotoxic cells by alloantigen-stimulated thymocytes, and 3) enhance the mitogenic response of thymocytes in the presence or absence of phytohemagglutinin. The uninduced control spleen cell supernatants contained variable but significantly less biologic activity within the difference assay systems. The principle enhancing activity (apparent m.w. 35,000 to 38,000) in each of the assay systems was found to be inseparable after a multiple step purification scheme involving ammonium sulfate precipitation, two-cycle gel filtration, hydroxylapatite chromatography, and hydrophobic chromatography on Phenyl Sepharose. These results indicate that either a single factor (designated thymocyte mitogenic factor) augments both humoral and cell-mediated immune responses or, alternatively, the different biologic activities are mediated by different factors that share a common core structure.

Ammonium Sulfate↗
PubMed1977

The xenogeneic effect. III. Induction of cell-mediated cytotoxicity by alloantigen-stimulated thymocytes in the presence of xenogeneic reconstitution factor.

A supernatant from human mixed leukocyte cultures (MLC), Xenogeneic Reconstitution Factor (XRF), was added to one-way murine MLC. This supernatant greatly enhances the generation of cytotoxic cells from C57BL/6 or DBA/2 responder thymocytes, as assayed by a standard 51Cr-release assay. This enhancement is shown 1) to be dependent upon the presence of BDF1 semi-allogeneic stimulator cells and 2) to result in the generation of cytotoxic cells that are specific for the H-2 type of the stimulating alloantigen. In some experiments, alloantigen-stimulated murine spleen cells, when cultured with XRF showed increased induction of cell-mediated cytotoxicity. These experiments show that XRF contains a cytotoxic-cell activating factor(s) that is functionally similar to those found in murine-derived supernatants, and that this factor can participate in the generation of cytotoxic cells from precursors present in the murine thymus.

Animals↗