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D Gallardo

Publications and source records attributed to D Gallardo.

40 records · Page 3Linked to original sources
PubMed1990

IL-1 and IL-4 as reciprocal regulators of IL-2 induced lymphocyte cytotoxicity.

Interleukin 4 (IL-4) suppresses the interleukin 2 (IL-2) induced lymphokine-activated killer (LAK) cell development from human peripheral blood mononuclear cells (PBMC). Suppression is observed at high (1,000 U ml-1) as well as low (10 U ml-1) concentrations of IL-2. IL-4 needs to be present at the beginning of the IL-2 culture to exert the suppressive effect. IL-4 also inhibits the development of CD25 (Tac) antigen on the PBMC cultured in IL-2. Interleukin 1 (IL-1) can reverse the suppressive effect of IL-4 on LAK induction when added at the early phase of the IL-2 culture. IL-1 enhances IL-2 induced LAK development, which may partially explain the reversion of IL-4 inhibition by IL-1. IL-1 also reverses the inhibitory effect of IL-4 on the development of CD25 antigen expression, although IL-1 alone does not enhance the induction of CD25 expression in PBMC cultured by IL-2. Furthermore, IL-4 suppresses IL-2 induced IL-1 production in PBMC. Thus, suppression of CD25 may be a pathway for the suppression of LAK induction. The expression of CD56 is not directly associated with the expression of LAK activity. IL-4, IL-1 or combination of the two cytokines has no effect on IL-2 induced expression of CD56. These results indicate that IL-4 has an antagonistic effect and IL-1 has a synergistic effect on IL-2-induced LAK development.

Antigens, Differentiation, T-Lymphocyte↗
PubMed1987

Altered antigenicity of human monoclonal antibodies derived from human-mouse heterohybridomas.

We have generated milligram quantities of human monoclonal antibodies (Hu-MAbs) in the ascites of pristane-primed nude mice injected with human-mouse heterohybridomas. After contaminating mouse immunoglobulins were removed by affinity chromatography, an enzyme immunosorbent assay (EIA) was used to measure the concentrations of human immunoglobulins. Ten different partially purified preparations were tested. The titration curves with all 5 IgG Hu-MAbs were unusual, reaching a plateau at a very low apparent maximum concentration of antibody. In contrast, the EIA yielded more usual titration curves and thus apparently more reliable estimates of the concentrations of 4 IgM and 1 IgA monoclonal antibodies. An analogous EIA for the quantitation of mouse IgG monoclonal antibodies also gave accurate estimates. To understand the nature of the discrepancy with human IgG, 5 Hu-MAbs of the 3 classes (2 IgG, 2 pentameric IgM and 1 IgA) were purified to homogeneity for a more detailed analysis. The inability to quantitate the human IgG monoclonal antibodies by EIA was not due to defective molecules, as shown by SDS polyacrylamide gel electrophoresis. The human IgG monoclonal antibodies were found to consist of intact heavy and light chains, as were the IgM and IgA antibodies. The possibility that the human IgG monoclonal antibodies differed antigenically from polyclonal IgG was explored by comparing the concentrations by EIA with the protein concentrations determined by absorbance at 280 nm. This analysis permitted a comparison of the detectability of antigenic determinants on Hu-MAbs with those on polyclonal Ig with goat antibodies to Ig or Ig subclass. The IgG monoclonal antibodies differed from polyclonal IgG in both their heavy and light chains. Goat antiserum monospecific for the gamma chain in fact underestimated the concentration by as much as one hundred-fold. IgM and IgA monoclonal antibodies were less antigenically distinct from their polyclonal counterparts even though their light chains were also underestimated, because goat monospecific antibodies were more efficient at recognizing their heavy chains. The molecular basis for the observed difference in antigenicity is not yet known. These findings have important implications for the analysis of the binding of IgG Hu-MAbs. A direct binding assay with the label directly conjugated to the Hu-MAb should be used in preference to an indirect assay with a labeled detecting antibody to maximize the sensitivity of the assay. The altered antigenicity of IgG Hu-MAbs may also imply decreased immunogenicity when they are given in vivo as carriers for radionuclides or cytotoxic antitumor materials.

Animals↗
PubMed1984

Rapid covalent coupling of proteins to cell surfaces: immunological characterization of viable protein-cell conjugates.

A rapid and efficient 2-step procedure is described for covalently attaching proteins to cell surfaces by using a heterobifunctional cross-linking agent, succinimidyl-4-(N-maleimidomethylcyclohexane)-1-carboxylate (SMCC). In the first step, protein is derivatized for about 30 min with a 1:1 (mole:mole) stoichiometric ratio of SMCC which creates 4-(N-maleimidomethylcyclohexane)-1-amidyl-protein (MCA-protein) covalent linkages with the primary amino groups of proteins. In the second step, cell-MCA-protein conjugates are rapidly prepared (in less than 30 min) by reacting MCA-protein with 'reduced' cells which have been pre-incubated (for about 1 h) with dithiothreitol (DTT). Stable protein-cell conjugates result from the covalent thioether linkages formed between the maleimido groups of the derivatized protein and the cell surface thiol groups created by DTT reduction. Protein-cell conjugates have been made in this way with several different proteins using several different types of cells. Such conjugates are characterized in this study by complement plus antibody-mediated cytotoxicity (CAMC) and by antibody-dependent cellular cytotoxicity (ADCC) with human effector lymphocytes. Because these protein-cell conjugates are shown to remain viable and to retain significant levels of coupled protein for at least 24 h in culture, they are potentially useful for probing various membrane-related phenomena such as the recognition of cell surface antigens by immune effector cells.

Animals↗