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Biomedical subjects

B M Stadler

Publications and source records attributed to B M Stadler.

At least 109 records · Page 6Linked to original sources
PubMed1986

Characterization of a monoclonal antibody directed against the biologically active site of human interleukin 1.

Human IL-1 was successfully used to produce an anti-IL-1 mAb. Anti-IL-1 (IgG2a) blocked IL-1-mediated thymocyte and fibroblast proliferation, but did not interfere with the biological effects of other lymphokines, such as IL-2 or IL-3. The antibody immunoprecipitated biosynthetically radiolabeled 33, 17, and 4 kD IL-1. An immunoadsorbent column yielded 20% of initial activity, and upon HPLC size-exclusion chromatography, affinity-purified IL-1 had a molecular mass of approximately 4 kD. These results provide first evidence of a monoclonal anti-IL-1 that reacts with different species of IL-1 and apparently binds to an epitope close to the active site of IL-1. Thus, anti-IL-1 IgG may be very helpful for further investigations of the molecular as well as biological characteristics of IL-1 and related mediators.

Animals↗
PubMed1986

Quantitative determination of total and specific human IgE with the use of monoclonal antibodies.

We have used two monoclonal antibodies (BS17 and Le27) that recognize two different epitopes of the constant region of human IgE in order to determine the total and specific IgE contained in human serum. Both types of assay are based on classic PRIST and RAST procedures and involve one or two monoclonal antibodies labeled with 125I iodine. The performance of these two assays were compared systematically with those obtained with a commercially available polyclonal tracer. We first investigated the 125I-labeling conditions for monoclonal antibodies. In our hands the best results were obtained by use of a tracer of specific radioactivity close to 10 microCi/micrograms. We have been able to demonstrate that as a consequence of its limited affinity, the radioactive tracer is always incompletely bound to the solid-phase IgE. Nonetheless, the sensitivity of the assay is comparable to that obtained with the polyclonal antibody, since less than 0.5 IU/ml can be measured by the PRIST procedure. In contrast, the dilution curves obtained in the RAST with the monoclonal antibodies are very different from those observed with the polyclonal tracer. In fact, these curves are strictly parallel to the dilution curve and to the standard curve derived from the PRIST method, thus indicating that the monoclonal antibodies recognize all IgE equally well regardless of the way in which they are bound to the solid phase. On the basis of this observation, we propose a quantitative assay of specific IgE with the PRIST standard curve as reference. Our results demonstrate that this approach is indeed possible if high-capacity, solid-phase as well as long incubation times are used.

Allergens↗
PubMed1986

Biological and biochemical characteristics of the basophil-like cell promoting activity (BaPA) and a human IL 3-like activity.

Two growth factors isolated from lectin-stimulated human mononuclear cells stimulate the long-term growth of metachromatically staining cells in human bone marrow cultures. One factor, termed basophil-like cell promoting activity, induces also differentiation of cells which morphologically and functionally resemble human basophils. A second factor, which in contrast to BaPA stimulates murine IL3-dependent cells, induces the growth of human metachromatically staining cells of an immature morphology with certain resemblance to mast cell-like cells. BaPA inhibits the growth of HL-60 cells, while IL3-like activity stimulates the growth of HL-60 cells. BaPA does not share biochemical similarities to other well-defined human growth factors, while the IL3-like activity has strong resemblance to pluripotential hemopoietic CSF.

Basophils↗
PubMed1986

Human IgE synthesis in vitro. Detection with monoclonal antibodies.

Using monoclonal anti-human IgE antibodies recognizing the D epsilon 1 or D epsilon 2 epitope, we have developed a sandwich radioimmunoassay (RIA) to determine IgE in human cell cultures. With the help of this assay, various methods of measuring an actual de novo IgE synthesis were compared. It was necessary to subtract performed IgE from released IgE in the culture supernatant and the IgE associated to the cultured cells, in order to determine a net IgE synthesis which would reflect de novo synthesized IgE. Using this differential in vitro IgE assessment, no net IgE synthesis could be demonstrated in culture conditions which lead to strong IgG synthesis. Actual net in vitro IgE antibody production was only found in approximately 30% of cell cultures from atopic donors. This spontaneous IgE synthesis did not correlate to the serum IgE levels of the patients. However, correlations were found between serum IgE levels and amount of performed, released or cell-associated IgE of the cultures.

Antibodies, Monoclonal↗
PubMed1986

Schistosome-derived inhibitory factor: an immunosuppressive agent preferentially active on T lymphocytes.

We have previously shown that schistosome-derived inhibitory factors (SDIF) inhibited lymphocyte proliferation and induced immunosuppression. Crude SDIF was purified by successive gel filtration and reverse-phase high-performance liquid chromatography. Purified SDIF preparations strongly inhibited the proliferation of different T cell line cells, while other cell lines (B cells, macrophages and fibroblasts) were almost not affected by SDIF. The inhibition of T cell proliferation by SDIF was not mediated through an Interleukin-2-dependent mechanism since both Interleukin-2-dependent and -independent T cells were inhibited. SDIF-activity was absorbed by cells in a time- and cell-number-dependent fashion at 4 degrees C, suggesting the existence of a possible receptor for SDIF. However, the difference in sensitivity to SDIF proliferation inhibition could not be attributed to the presence or absence of this receptor since cells from SDIF-sensitive and SDIF-resistant cell lines absorbed SDIF activity in the same way.

Absorption↗
PubMed1986

Histamine release from human leukocytes by anti-IgE antibodies: influence of multiple or single epitope recognition.

Upon comparing several polyclonal anti-IgE antisera (specific for the Fc epsilon fragment) for their ability to release histamine, we observed a marked heterogeneity of response for the same antiserum in different cell donors as well as for different antisera in the same cell donor. Additional studies with monoclonal antibodies directed against various epitopes of the D1 and D2 regions revealed several possible explanations: broad specificity of the polyclonal antisera, lack of availability of some epitopes on the cell-bound IgE (which were available on soluble IgE), microheterogeneity among IgE molecules from different patients, and failure of some donors' cells to respond to anti-IgE antibodies. We conclude that selection of appropriate antisera is an important consideration in comparing IgE-mediated histamine release among individuals.

Antibodies, Anti-Idiotypic↗
PubMed1985

Factors influencing human IgE synthesis in vitro and in vivo.

Several pitfalls may affect studies on human IgE synthesis in vitro. In this paper, the requirement for stringent specificity of the anti-IgE antibodies used and for assessment not only of IgE detectable in culture supernatants but also as cell-associated IgE is emphasized. The use of cycloheximide-treated cultures as controls also leaves wishes open. Activated, human T cells and T cell hybridomas produce IgE-binding factors, which may be detected by a sensitive in vitro test and which may apparently also become the endeavour of synthesis by molecular biological techniques. Although the evidence available in rodents for the role of IgE-binding factors in modulating IgE synthesis has not yet been fully reproduced by us in man, the fact that classical IgE-enhancing procedures in rodents (e.g. radiotherapy, T cell suppression) also affect IgE production in man leads to believe that similar immunoregulation mechanisms apply to various mammalian species studied so far.

Antibody Formation↗
PubMed1985

Distinction of the human basophil promoting activity from human interleukin-3.

Lectin-stimulated supernatants of human peripheral blood leukocytes (PBL) contained an activity that maintained the long-term growth of murine interleukin-3 (IL-3) dependent cell lines such as the 32 Dcl line. We compared this human IL-3 activity with the basophil promoting activity (BaPA) recently described by us. Besides lectin-stimulated PBL supernatants that contained both BaPa and IL-3, we found that BaPA was produced mainly by E rosette-positive T cells or the human Mo T cell line, while IL-3 was produced by human T hybridomas and, more interestingly, by human macrophage cell lines. Based on molecular weight (BaPA: 20-30 kilodaltons, IL-3: 15-19 kilodaltons) as well as on molecular charge (BaPA: pI = 6.1-7.5, IL-3: 4.9-6.2), we concluded that human IL-3 and BaPA are distinct factors.

Basophils↗
PubMed1984

Interleukin 2-independent stimulation of rabbit chondrocyte collagenase and prostaglandin E2 production by an interleukin 1-like factor.

In the present study, interleukin 1 (IL 1)-containing media from different sources, namely a murine macrophage cell line (P388D1), rabbit peritoneal macrophages, and human peripheral blood mononuclear cells, were compared for their effect on thymocyte proliferation and on collagenase and PGE2 secretion by chondrocytes. A high correlation was found between the enhancement of thymocyte proliferation and the induction of collagenase and PGE2 secretion by chondrocytes. Furthermore, a highly purified IL 1-like factor, namely mononuclear cell factor (MCF) was also active on chondrocytes. The addition of highly purified IL 2 to rabbit chondrocytes had no effect on collagenase and PGE2 secretion induced by IL 1-containing media. Our findings suggest that the factor which induced collagenase and PGE2 secretion by rabbit chondrocytes was an IL 1-like factor. Thus, collagenase secretion by chondrocytes may be used as an IL 2-insensitive assay for the detection of IL 1-like factors.

Animals↗
PubMed1984

Isolation of human mononuclear cell subsets by counterflow centrifugal elutriation (CCE). II. Functional properties of B-lymphocyte-, T-lymphocyte-, and monocyte-enriched fractions.

Counterflow centrifugal elutriation (CCE), a technique which separates cells by size and density, was used to separate human peripheral blood mononuclear cells into fractions enriched for T lymphocytes, B lymphocytes, and monocytes. These morphologically and phenotypically distinct fractions were analyzed for their ability to respond in several functional assays. B-Cell-enriched fractions devoid of monocytes did not proliferate nor produce significant quantities of lymphokines in response to antigens. These B cells did proliferate to anti-IgM antibodies but not to anti-IgD antibodies. B-Cell fractions served as stimulators of the autologous mixed-lymphocyte reaction (AMLR). T-Lymphocyte fractions were unable to respond to antigen challenge, but both proliferation and lymphokine production could be restored by the addition of monocytes. Monocyte fractions produced PGE2, displayed chemotaxis, and functioned as stimulators in the AMLR. Thus, CCE appears to be a useful technique for reproducibly obtaining highly enriched subsets of human peripheral blood mononuclear cells with unique phenotypic and functional properties. These isolated populations can consequently be used to identify the independent and collaborative roles of the cells in immunological events.

Antibodies, Anti-Idiotypic↗
PubMed1984

Transcription and translation dependent induction of interleukin 2 (IL-2) and IL-2 receptors.

Our previous findings on the expression of Interleukin 2 (IL-2) receptors have shown that under the influence of certain drugs, it was possible to dissociate IL-2 production from the appearance of IL-2 receptors on human peripheral blood lymphocytes (PBL). Therefore, we studied whether the induction of IL-2 receptors were transcription- or translation-dependent. Actinomycin D and puromycin inhibited the appearance of IL-2 receptors as well as the production of IL-2. Furthermore, both drugs also inhibited human PBL to enter the G1 phase of the cell cycle. Thus, these data suggested that both IL-2 production and the appearance of IL-2 receptors were dependent on transcription and translation in non-resting cells.

Adult↗
PubMed1983

Factor-dependent in vitro growth of human normal bone marrow-derived basophil-like cells.

A factor(s) present in supernatants from lectin-stimulated peripheral blood mononuclear cells promoted the production of basophil-like cells in liquid cultures of normal human bone marrow cells. The cultured basophil-like cells had lobulated or round nuclei, and the cytoplasmic granules stained metachromatically with toluidine blue and azurophilic with Giemsa. 20% of the metachromatically staining cells were peroxidase positive but not positive for nonspecific esterase. The histamine content was 0.5-2 pg/cell. The basophil-like cells released histamine upon challenge with calcium ionophore A23187 but not with compound 48/80. They also released histamine with anti-IgE when passively sensitized with human myeloma IgE. The development of basophil-like cells was promoted in a dose-dependent fashion by a factor(s) in the conditioned medium. Blocking of cell proliferation with hydroxyurea or X irradiation inhibited the development of basophil-like cells. The production of the factor was dependent on the presence of T cells. The factor was different from interleukin 2 and its molecular weight was estimated to be 25,000-40,000 by gel filtration on a Sephacryl S-200 column. Thus, human basophil-like cells derived from normal bone marrow cells can grow and differentiate in vitro under the regulation of T cells.

Basophils↗
PubMed1983

Characteristics of an epidermal cell thymocyte-activating factor (ETAF) produced by human epidermal cells and a human squamous cell carcinoma cell line.

In this study we show that both cultured normal human epidermal cells (EC) and a human squamous cell carcinoma (SCC) cell line produce a thymocyte-activating factor (ETAF). EC-ETAF and SCC-ETAF both have a Mr of 15,000 and were eluted from chromatofocusing at the same isoelectric points of 7.2, 5.8, and 5.0. Both activities were maintained at alkaline pH and were destroyed at temperatures above 60 degrees C. In addition to stimulating thymocyte proliferation, human ETAF exhibited a variety of other pertinent biologic activities. Although EC-ETAF or SCC-ETAF by themselves exhibited no T-cell growth factor activity, both ETAF preparations enhanced Interleukin 2 production by cultured human peripheral blood lymphocytes when stimulated with polyclonal T-cells stimulants (Concanavalin A and phorbol myristate acetate). Human ETAF also was chemotactic for rabbit polymorphonuclear leukocytes and was directly mitogenic for cultured human dermal fibroblasts. Injection of human ETAF into C3H/HeJ mice, resulted in inducing serum amyloid A (SAA) production by murine hepatocytes. The thymocyte growth-enhancing activity, the fibroblast-stimulating activity, and the SAA-inducing capacity of ETAF all coeluted off AcA54 gel. These biologic as well as biochemical properties of human keratinocyte-derived ETAF are identical with those of human macrophage-derived Interleukin 1. The ability of keratinocytes to release an immunomodulating factor with such diverse consequences may play an important role in normal wound healing and in diseases involving epithelial tissues.

Carcinoma, Squamous Cell↗
PubMed1983

Cell cycle associated changes in histamine release from rat basophilic leukemia cells separated by counterflow centrifugal elutriation.

This study evaluated histamine release from cells at different stages of the cell cycle. Cells from the cloned rat basophilic leukemia subline (RBL-2H3) were fractionated by counterflow elutriation according to size and density. The smallest cells were predominantly in the G1 phase of the cell cycle. These cells contained the least histamine and after IgE-mediated triggering released the lowest fraction of their total histamine. In contrast cells in the S, G2, and M stages were larger, contained more histamine and released more of their histamine after activation. When G1 stage cells were recultured, there was an increase in cell size, in histamine content and histamine release. Therefore, there is heterogeneity in the capacity of cells for IgE-mediated triggering at different stages in the cell cycle, with optimal release from the more mature cells.

Animals↗
PubMed1983

Interleukin 2 dependence of human natural killer (NK) cell activity.

When purified populations of human natural killer (NK) cells were tested for cytotoxic activity in the presence of partially purified preparations of human interleukin 2 (IL 2), a definite, dose-dependent linear increase in reactivity was observed. To determine whether such augmentation by IL 2 might reflect an important aspect of the physiologic regulation of NK activity, we examined the effects of monoclonal antibodies against human IL 2 on spontaneous NK activity. The presence of such antibodies during the 4-hr cytotoxicity assay resulted in significant inhibition of NK activity, and when the NK cells were pretreated for 16 to 20 hr with anti-IL 2, little or no activity remained. These data suggest that the spontaneous cytotoxic activity of NK cells is dependent on their continued exposure to IL 2. The reduction in NK activity resulting from treatment with anti-IL 2 could be at least partially restored by exposure to only low amounts of partially purified IL 2. These data have provided the basis for formulating a novel model of NK cell activation.

Animals↗