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Biomedical subjects

B M Stadler

Publications and source records attributed to B M Stadler.

At least 127 records · Page 7Linked to original sources
PubMed1982

Synthesis of biologically active interleukin 2 by Xenopus oocytes in response to poly(A)-RNA from a gibbon T-cell line.

The poly(A)-containing RNA isolated from a gibbon lymphosarcoma cell line, MLA144, known to release Interleukin 2 (IL2) was microinjected into Xenopus oocytes. The incubation medium from injected oocytes stimulated the DNA synthesis of an IL2-dependent cell culture and also maintained its viability. DNA-stimulating activity appeared in the oocyte incubation medium 6 h after injection and continued to accumulate for at least 96 h. Thymidine incorporation by the IL2-dependent cells is proportional to the concentration of oocyte incubation medium added and the IL2 activity produced by the oocytes is proportional to the amount of poly(A)-RNA injected. Incubation medium of oocytes injected with RNA from another T-cell line, 6G1, which does not produce IL2, did not contain DNA-stimulating activity. The same assay also showed that the IL2 mRNA sedimented at 14-16 S in a nondenaturing sucrose gradient. A series of monoclonal antibodies prepared against human IL2 neutralized the DNA-stimulating activity released by the injected oocytes to the same extent as they neutralized human IL2.

Animals↗
PubMed1982

Cellular origin of interleukin 2 (IL 2) in man: evidence for stimulus-restricted IL 2 production by T4+ and T8+ T lymphocytes.

The human T cell subset(s) responsible for production of interleukin 2 (IL 2) was investigated in the present study. For this purpose, highly purified T4+ and T8+ T lymphocytes were stimulated with mitogens and alloantigens. Subsequently, culture supernatants were analyzed for IL 2 activity in each of two assay systems: 1) proliferation of long-term T cell lines and 2) induction of cytotoxic effector cells from a resting T8+ population incubated in MLC. Mitogen stimulation led to secretion of equivalent amounts of IL 2 from both the major T cell subsets; in contrast, after allogeneic activation, IL 2 was produced predominantly from the T4+ subset. The stimulus dependency of IL 2 production suggests that the individual functional repertoires of T4+ and T8+ T cell subsets may be linked to unique surface receptors and/or interaction molecules involved in cell triggering rather than a restricted capacity to produce lymphokine.

Antigens, Surface↗
PubMed1982

Lymphokines: their role in lymphocyte responses. Properties of interleukin 1.

Interleukin 1, or IL 1, otherwise known as lymphocyte-activating factor, is a macrophage-derived 12,000- to 15,000-dalton polypeptide. Isoelectric focusing of human IL 1 reveals three peaks at pI's of 5.2, 6.0 and 6.9 respectively. IL 1 can be depleted of lymphocyte-derived IL 2 by SP-Sephadex chromatography. IL 1 augments the mitogenic response of PNA- Lyt 1+ thymocytes, and promotes thymocyte helper functions and B cell antibody production. IL 1 induces stable E rosette formation and the production of lymphokines such as T cell growth factor (IL 2) by peripheral T lymphocytes. Others have shown that IL 1 or closely related factors also stimulate hypothalamic cells to induce fever; induce in vitro fibroblast growth, prostaglandin, and collagenase production; and stimulate hepatocytes to produce acute phase proteins such as serum amyloid A. Murine epidermal cells also produce a 15,000-dalton factor that is mitogenic for thymocytes and may be similar to IL 1. We have recently hybridized spleen cells from mice sensitized with partially purified human IL 1 with a myeloma cell line. Clones have been isolated that produce supernatants that partially inhibit the thymocyte proliferative response to IL 1 but not the T cell growth factor activity of IL 2. Should these hybridoma products prove to be monoclonal anti-IL 1 antibodies, they will facilitate the further purification and characterization of IL 1.

Animals↗
PubMed1982

Murine epidermal cell-derived thymocyte-activating factor resembles murine interleukin 1.

We have previously described an epidermal cell-derived thymocyte-activating factor (ETAF), which is produced by the murine PAM 212 keratinocyte cell line. ETAF appeared to be similar to macrophage-derived interleukin 1 (IL 1) in its biologic activities and biochemical characteristics. Both IL 1 and ETAF augment thymocyte proliferation, enhance lymphocyte production of interleukin 2 (IL 2), and are 15,000 m.w. polypeptides that are stable at pH 4 to 11 and from -70 degrees C to 60 degrees C. In this study we describe a quantitative microassay to obtain standardized assessment of ETAF activity, which enabled us to further define the characteristics of ETAF and its relationship to IL 1. Just as stimulated macrophages produce more IL 1 activity, Pam 212 keratinocyte production of ETAF activity was increased by stimulation with lipopolysaccharide (LPS) or silica. Increased levels were also obtained by mechanical disruption of confluent monolayers of keratinocytes and by blocking proliferation of the Pam 212 cells with hydroxyurea at the G1/S interphase. These observations in conjunction with a concomitant decrease in keratinocyte viability suggest that "injurious" stimuli that prolong the G1 phase of the cell cycle factor ETAF production. ETAF, like murine IL 1, has an isoelectric point of 5.2. The same subpopulations of PNA-thymocytes that respond to PHA and IL 1 are responsible for the enhanced proliferative response to ETAF. Furthermore, as in the case of IL 1, PNA- Lyt-2- thymocytes were most responsive to ETAF, but not PNA+ LYt-2+ thymocytes. Finally, ETAF activity, like IL 1, appears to be a mitogenic signal for fibroblasts. Although produced by different cell types, these observations continue to support the view that ETAF may be identical or closely related to IL 1.

Animals↗
PubMed1982

Monoclonal antibody against human interleukin 2 (IL 2). I. Purification of IL 2 for the production of monoclonal antibodies.

Human interleukin 2 (IL 2) was produced under serum-free conditions by stimulating mononuclear cells with concanavalin A (Con A) in the presence of phorbol myristate acetate (PMA) and hydroxyurea. The IL 2 was partially purified by sequential chromatography by using phenyl-Sepharose, DEAE Sephacel, and AcA 54 gel filtration. This partially purified material was used to immunize BALB/c mice. After immunization with a total of 48,000 U (spec. act. approximately 10(5) units/mg protein), the spleen cells were adoptively transferred into x-irradiated syngeneic mice and the animals were boosted with another 12,000 U of IL 2. Four days later their spleen cells were hybridized with plasmacytoma cells. Supernatants of the hybridoma cultures were screened for their capacity to inhibit the IL 2-induced proliferation of the CT6 cell line. After expansion and cloning eight different lines were selected for ascitic antibody production. The monoclonal antibodies inhibited the proliferation of the IL 2-dependent cell line in response to either human crude or purified IL 2, as well as rat and mouse IL 2. However, these anti-IL 2 antibodies did not inhibit the proliferation of human T cell lines capable of producing IL 2. Monoclonal antibodies coupled to Sepharose 4B absorbed IL 2 crude culture supernatant, confirming that they react directly with IL 2. The absorbed IL 2 could, for the most part, be eluted by using sodium dodecyl sulfate, thus providing a means for further immunoaffinity purification of IL 2.

Animals↗
PubMed1981

Flow-cytometric analysis of human basophil degranulation. II. Degranulation induced by anti-IgE, anti-IgG4 and the calcium ionophore A23187.

Quantification of human basophil degranulation induced by anti-IgE, anti-IgG4, and by ionophore was performed using a flow-cytometric system. It was shown that these antibodies and ionophore can degranulate basophils in a dose-dependent manner, and that there is a wide variation in the response of basophils obtained from different individuals to these stimuli. A significant correlation was observed between the degree of degranulation induced by anti-IgE and anti-IgG4, while this was not the case for anti-IgE and ionophore. It was also shown that IgG4 myeloma protein can passively sensitive basophils. In general, degranulating efficacy was in the order of ionophore greater than anti-IgE greater than anti-IgG4, both in allergic and non-allergic individuals.

Anti-Bacterial Agents↗
PubMed1981

Flow-cytometric analysis of human basophil degranulation. I. Quantification of human basophils and their degranulation by flow-cytometry.

Quantification of human basophils and their degranulation were performed using a flow-cytometric system. It was shown that the number of basophils among total leukocytes, before and after the effect of degranulating agents, can be obtained rapidly and reproducibly, following alcian blue staining. Dose-response curves of degranulation by anti-IgE and anti-IgG4 were studied, and it was shown that these antibodies can induce basophil degranulation in a Ca++ dependent manner. It was also shown that degranulation is completed within 10 min. Values obtained by flow-cytometry and microscopic evaluation, as well as comparison made between percent basophil degranulation and percent histamine release, agreed well with each other in our system. Flow-cytometric analysis of human basophil degranulation provides a new reliable method to assess in vitro the morphological basis of immediate hypersensitivity.

Antibodies, Anti-Idiotypic↗
PubMed1981

Epidermal cell (keratinocyte)-derived thymocyte-activating factor (ETAF).

In order to determine whether keratinocytes play a role in the modulation of the immune response, we investigated the murine keratinocyte cell line Pam 212. In culture these cells generate a substance with a biologic activity that greatly enhances phytohemagglutinin-induced thymocyte proliferation. We have, therefore, called this substance epidermal cell thymocyte-activating factor (ETAF). This keratinocyte-derived supernatant activity is mainly produced at the onset of the logarithmic growth phase and is directly mitogenic for murine thymocytes. Although ETAF by itself exhibits no T cell growth factor activity, ETAF enhances Interleukin 2 production by mitogen-stimulated murine spleen cells. Murine ETAF is not genetically restricted and lacks species specificity since it decreases lectin-induced proliferation of human peripheral blood lymphocytes (as well as murine spleen cells) and also enhances the production of human Interleukin 2. The factor has a m.w. between 15,000 and 25,000 as determined by gel filtration and elutes as a single peak from anion exchange chromatography columns. The activity is maintained mainly at alkaline pH and is rapidly destroyed at temperatures above 60 degrees C. These observations suggest that epidermal cells may interact with the immune system by elaborating nonspecific factors that modulate lymphocyte proliferation and augment lymphokine production.

Animals↗
PubMed1980

Determination of rat mast cells by flow-cytometry.

Counting of rat mast cells and assessment of degranulation were performed by flow-cytometry using a cytograph. It was shown that the number of mast cells in a total cell population may be obtained rapidly and reproducibly following toluidine blue staining. A dose-response for degranulation by compound 48/80 was performed and it was shown that azure A staining (in 40% sucrose) discriminates degranulated from non-degranulated mast cells, while trypan blue staining discriminates between cytotoxic and non-cytotoxic degranulation. Values obtained by flow-cytometry and microscopic evaluation were in good agreement; assessment by flow-cytometry provides a convenient new approach to counting mast cells and assessing their degree of degranulation.

Animals↗
PubMed1980

Flow-cytofluorometric determination of active and total rosettes.

A flow-cytofluorometric technique is described for determining active and total rosettes formed by sheep red blood cells with human peripheral blood lymphocytes. This technique correlates well with the microscopic determination of active rosettes, when these are defined as three or more erythrocytes per lymphocyte, and with the determination of total rosettes, when these are defined as four or more erythrocytes per lymphocyte. In the small group of tumor patients tested, the observed decrease in capacity to form active rosettes was found to be dependent on the individual sheep red blood cells used, while the levels of total rosettes were not affected by individual SRBC's and were not significantly different from those of normal blood donors. Flow-cytometry is a rapid, reproducible and objective technique, which permits better measurement than microscopy of total and active rosette levels in both normal donors and cancer patients.

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↗
PubMed1978

Cytofluorometric analysis of macrophages activated in vivo or in vitro.

The relative RNA content of single macrophages was measured by cytofluorometry after differential staining of cellular DNA and RNA with the metachromatic fluorescent dye acridine orange. This method allowed the differentiation of two major groups in the adherent macrophage population differing in their RNA content. After in vivo stimulation of mice (by injection of thioglycollate medium) or guinea pigs (by injection of oil), an increasing percentage of macrophages possessing a high RNA content is recovered. In vitro stimulation of macrophage cultures with lipopolysaccharide had the same enhancing effect on cellular RNA content. Cytofluorometric measurement of RNA content may possibly become an efficient and rapid method of measuring macrophage activation.

Acridines↗
PubMedDate not supplied

Mimotope and anti-idiotypic vaccines to induce an anti-IgE response.

We have defined epitopes on human IgE by screening different phage display random peptide libraries with a monoclonal anti-IgE antibody termed BSW17. The selected mimotopes and epitopes within the Cepsilon3 and Cepsilon4 region of IgE induced antibodies that were nonanaphylactogenic and had biological activity similar to BSW17. The chemically synthesized and KLH-coupled IgE epitopes or mimotopes were used to induce an anti-IgE response in rhesus monkeys. The immunized rhesus monkeys were subsequently protected in a PCA test when sensitized with human IgE and triggered with the corresponding allergen. Furthermore, using the same monoclonal anti-IgE antibody, we also generated an anti-idiotypic antibody that showed sequence homology with the IgE epitope in the Cepsilon3 domain. This anti-idiotypic antibody as well as the mimotopes were then used in a mouse model to induce orally an anti-IgE immune response. For this purpose mice were fed by intragastric gavages with bacteriophages displaying the small IgE-homologous structures. Orally immunized mice produced serum anti-IgE antibodies that were inhibited by BSW17 suggesting that it may be possible to induce a systemic anti-IgE response orally.

Animals↗