[In vitro diagnosis of penicillin allergy by dot immunobinding assay].
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Anaphylaxis↗
Biomedical subjects
B M Stadler
Publications and source records attributed to B M Stadler.
Differential expression of cytokine mRNAs in human cell lines.
We investigated the expression of several cytokine genes known to be differentially expressed in human mononuclear cells depending on the way of stimulation. In order to investigate cytokine gene expression in cell lines we stimulated peripheral blood mononuclear cells, the human cell lines UBC 5637, Wil-2, K562, U937 and Jurkat with PHA or a phorbol ester. The patterns of cytokine expression were assessed at 8 and 20 hours after stimulation by Northern blot analysis. Our data show that after stimulation each cell line expressed its own pattern of cytokine mRNA levels. Additionally, superinduction with cycloheximide revealed no change of a stimulation dependent pattern. Furthermore, we found that each cytokine has its own kinetic suggesting that signal transduction may be followed by cell type specific intracellular mechanisms.
Biological Factors↗
A dot immunobinding assay for penicillin specific antibodies: allergic diagnosis and prediction.
A dot immunobinding assay was used to determine benzylpenicillin (BPO) specific IgE and IgG antibodies. Sera from 39 patients who had a positive history of penicillin allergy and the same number of sera from normal donors without penicillin-allergic history were assessed. The results showed that the mean value of BPO-specific IgE in the group of patients was 3.6 ng/ml while in the group of normal donors it was 0.38 ng/ml. BPO-specific IgE values of 35 samples in the group of patients were greater than 2 ng/ml. In contrast, only one sample in the group of normal donors amounted to 2 ng/ml. Thus 2 ng/ml was arbitrarily chosen to discriminate between normal and patients' sera. Based on this assumption the positive rate is 89.7 percent; the false positive rate would only be 2.6 percent. Furthermore, in the group of patients, the mean value of BPO-specific IgG was 49.1 ng/ml, while in the group of normal donors it was 7.4 ng/ml. However, due to the large variability of serum IgG levels it was not possible to determine a BPO-specific IgG concentration useful for diagnosis or prediction of penicillin allergy.
Adolescent↗
Stimulation of tumor cell growth in humans by a mononuclear cell-derived factor.
Serum-free supernatants of peripheral blood mononuclear cell cultures significantly stimulated [3H]thymidine incorporation of human hematopoietic and nonhematopoietic tumor cell lines. For assay we used a low number of tumor cells per well and medium enriched with dithiothreitol-treated fetal calf serum. The growth-stimulatory activity was detected in the supernatant of peripheral blood mononuclear cell culture within the first 24 h and decreased thereafter. Treatment of mononuclear cells with OKT3 monoclonal antibodies and rabbit complement decreased only moderately the factor production while treatment with anti-Leu-M2 and rabbit complement decreased it significantly. Supernatants of concanavalin A-stimulated peripheral blood mononuclear cell cultures enhanced nonsignificantly [3H]thymidine incorporation by tumor cell cultures unless antibodies against tumor necrosis factor alpha and gamma-interferon were added to the supernatants. Growth-stimulatory activity was heat inactivated partially at 60 degrees C and totally at 80 degrees C. It was abolished at pH 2.5 within 2 h as well as by treatment with dithiothreitol and partially lost by dialysis.
Antibodies, Monoclonal↗
Characterization of a human basophil-like cell promoting activity.
Biologic and biochemical properties of a human basophil-like cell promoting activity (BaPA), which induces growth of metachromatically staining cells from normal bone marrow cells in a liquid culture system have been examined. In order to study this T cell factor, an assay was developed based on the intracellular histamine content of the cultured human bone marrow cells. Many lymphokines, including granulocyte-macrophage colony-stimulating factor, granulocyte colony-stimulating factor, interleukin 1 alpha and 1 beta, interleukin 2, and interferon-alpha and gamma, did not exhibit any significant activity in the assay. By employing this assay, BaPA was purified approximately 500-fold from lectin-stimulated spleen cell-conditioned medium. BaPA has a molecular weight of 23,000 on high performance liquid chromatography gel filtration and displays isoelectric points between 5.8 and 7.3. It is heat stable up to 80 degrees C for 30 min and resistant to 6 M guanidine hydrochloride, whereas it is rather sensitive to sulfhydryl reagents. BaPA has no stimulating activity on mouse bone marrow cells.
Basophils↗
Induction of human in vitro IgE synthesis via stimulation by anti-CD3 antibody.
Mitogens such as pokeweed mitogen, Staphylococcus aureus Cowan I, concanavalin A and phytohemagglutinin and monoclonal anti-CD antibodies were examined for their capacity to induce IgE synthesis by peripheral blood leukocytes obtained from both atopic and nonatopic individuals. While lectins failed to induce IgE synthesis, monoclonal anti-CD3 antibodies were very potent stimuli for the induction of human in vitro IgE synthesis, possibly due to the activation of T cells. Activation via CD4 or CD8 molecules by OKT4 or OKT8 antibodies did not lead to a T cell-dependent modulation of IgE synthesis. PWM acted in synergy with the enhancing effect of anti-CD3 antibodies for IgE synthesis. In vitro IgG production was less affected. These results indicate that activation of T cell via CD3 molecules may be important in the regulation of IgE immune responses in man. Furthermore, the successful induction of IgE synthesis by anti-CD3 antibody in unfractionated peripheral blood leukocytes culture provides a simple model for investigation of human IgE regulatory mechanism in vitro.
Antibodies, Monoclonal↗
Stimulation-dependent lymphokine mRNA levels in human mononuclear cells.
Steady-state mRNA levels for lymphokines were studied in human peripheral blood mononuclear cells stimulated by various agents. The pattern of mRNA levels differed according to the way in which cells were stimulated or activated. Stimulation by anti-CD3 antibodies led to increased mRNA levels for interferon-gamma but only extremely low levels of interleukin 2 mRNA were detected. In order to observe levels of interleukin 2 mRNA, it was necessary to use phorbol myristate acetate (PMA) in addition to either lectins or anti-CD3 antibodies. PMA by itself was already able to induce high levels of mRNA for granulocyte macrophage colony-stimulating factor. Most interestingly, different patterns of lymphokine production were obtained with stimuli yielding the same number of activated cells and the same ratio of activated CD4 and CD8 cells. Thus, different T cell stimuli induced distinct patterns of lymphokine mRNA levels.
Antigens, Differentiation, T-Lymphocyte↗
Effect of recombinant human interleukin 4 on spontaneous in vitro human IgE synthesis.
Recombinant human interleukin 4 (IL 4) alone enhanced the spontaneous IgE synthesis in cultures of peripheral blood leukocytes (PBL) from atopic patients as well as from nonatopic individuals, suggesting the existence of preactivated PBL sensitive for IL 4. Preactivated cells were also obtained by stimulation with Staphylococcus aureus strain Cowan I (SAC). However, co-stimulation of PBL by IL 4 with SAC or anti-IgM antibody and pokeweed mitogen did not result in an enhanced IgE synthesis. Optimal IL 4 concentrations for the induction of IgE synthesis coincided with optimal proliferative responses in PBL. The effect of IL 4 was not isotype specific, and in terms of protein even more IgG and IgM antibodies were formed. The effect of IL 4 on IgE synthesis was counteracted by very low concentrations of interferon-gamma (IFN-gamma), suggesting that both IL 4 and IFN-gamma might be decisive cytokines for the human in vitro IgE synthesis.
Cells, Cultured↗
Production of hemopoietic growth factors by bone tissue and bone cells in culture.
This study was carried out to determine whether bone might be a source of hemopoietic growth factors. Both neonatal murine calvaria and primary cultures of cells isolated from calvaria released, upon stimulation with lipopolysaccharide, an activity that stimulated the growth of the interleukin (IL) 3-dependent cell lines, 32D cl, 123, and NSF 60. Upon gel filtration, this activity eluted with a molecular weight of 30,000 kDa. Further characterization, however, revealed that the major activity in conditioned medium was not IL 3. Activity was absorbed by DEAE-Sephacel at low salt concentration, whereas IL 3 does not adhere. Furthermore, an IL 3-specific antiserum did not neutralize the activity from cells and only partly neutralized the activity generated by whole calvaria. After gel filtration, the 30-kDa activity stimulated the growth of very large colonies in semisolid medium consisting mainly of granulocytes with the remainder being macrophages. No colony types belonging to other hemopoietic lineages were found, indicating, again, that the activity was not identical to IL 3. Subsequently, conditioned medium was fractionated by hydrophobic chromatography on Phenyl-Sepharose CL-4B, yielding two peaks of activity. Neutralization of activity with antisera to granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL 3 and use of colony assays showed that medium conditioned by whole calvaria contained GM-CSF and granulocyte CSF (G-CSF) in similar amounts together with a little IL 3, and medium conditioned with calvaria cells contained GM-CSF and little G-CSF. We conclude that bone releases hemopoietic growth factors that could contribute both to hemopoiesis and to the recruitment of osteoclasts from progenitors resident in the adjacent marrow.
Animals↗
Anti-amiodarone antibodies: detection and relationship to the development of side effects.
PURPOSE: It has become evident in the past few years that amiodarone, a powerful antiarrhythmic agent, induces considerable side effects. These may be due to an amiodarone-elicited lipid storage disease and to the iodine content of amiodarone, but might also be causally related to amiodarone-induced immune reactions. The latter possibility prompted us to develop a sensitive anti-amiodarone antibody detection assay based on the immunodot technique. PATIENTS AND METHODS: Sera were obtained from 10 untreated control subjects and 33 patients receiving amiodarone. Using serum dilutions of 1:500 and 1:1,000, the lower detection limit was 0.3 microgram/ml of anti-amiodarone antibodies as calculated from a simultaneously performed IgG standard curve. RESULTS: Screening of sera from the untreated control subjects and amiodarone-treated patients revealed that the untreated subjects had no anti-amiodarone antibodies, that only one of 16 patients without clinical side effects had elevated anti-amiodarone antibodies, but that seven of 12 patients with amiodarone-induced thyroid disease and four of five patients with other side effects had elevated anti-amiodarone antibody titers (1.2 to 2.5 micrograms/ml). The combined evaluation of anti-amiodarone antibody titers and cumulative dose was found to be a highly reliable indicator of side effects, as all patients with more than 100-g cumulative dose of amiodarone and more than 0.6 microgram/ml of anti-amiodarone antibodies had side effects. CONCLUSION: The detection of anti-amiodarone antibodies in patients with amiodarone-elicited side effects underscores the possible contribution of immunologic reactions to the development of certain side effects.
Adult↗
Further biochemical characterization of human basophil-like cell-promoting activity and its distinction from human interleukin-3-like activity.
Supernatants from lectin-stimulated human peripheral blood mononuclear cells contained two activities; basophil-like cell-promoting activity (BaPA) and a growth factor for murine interleukin-3-dependent cell lines (IL-3-like activity). BaPA had a MW of 24,000 and a pI of 6.0-7.5, while human IL-3-like activity had a MW of 18,000 and a pI of 5.0-5.9. Beside the biochemical differences between the two activities, supernatants from a T cell line were found that contained BaPA and GM-CSF but no IL-3-like activity. These data suggest that BaPA and IL-3-like activity are two different molecular entities.
Ammonium Sulfate↗
Lymphokine production by human peripheral blood lymphocytes: analysis by in situ hybridization.
Lymphokine production in human peripheral blood mononuclear cells (PBMNCs) was investigated by in situ hybridization with 35S-labelled RNA probes coding for interleukin 2 (IL-2), gamma-interferon (gamma-IFN) and granulocyte/macrophage colony stimulating factor (GM-CSF). The proportion of PBMNCs expressing lymphokine mRNA varied according to the stimuli used and followed similar patterns to those previously observed by Northern blot analysis. Only a relatively low proportion of PBMNCs expressed lymphokine mRNA after stimulation for a given time, and this seems not to be due to the heterogeneity of T cells.
Colony-Stimulating Factors↗
Biochemical characterization of the human basophil-promoting activity.
Human T cells produce a factor that induces growth of metachromatically staining cells in human bone marrow cultures. These cultured cells contain metachromatically staining granules and release histamine upon triggering with IgE and anti-IgE antibody. Based on morphological criteria, these cultured cells were termed basophil-like cells. We have generated a human T hybridoma which produces this basophil-like cell-promoting activity (BaPA). BaPA has a molecular weight of approximately 20 kilodaltons and isoelectric points between pH 5.8 and 7.5, with a major peak at pH 7. BaPA is of protein nature and can be clearly separated from interleukin-1 (IL-1), IL-2, interferons, GM-CSF and M-CSF. BaPA is also clearly different from a human IL-3-like activity which by itself can induce growth of metachromatically staining cells containing lower histamine levels than the cells cultured in the presence of BaPA. The growth of human basophil/mast cell-like cells can furthermore be enhanced if human bone marrow cells are cultured in the presence of fibroblast feeder cells.
Bone Marrow Cells↗
Human T hybridoma-derived immunoglobulin-binding factors.
A human T hybridoma was generated lacking demonstrable low-affinity IgE surface receptors. This cell line produces spontaneously immunoglobulin-binding factors. The factors have an affinity for human IgE and IgG as well as for IgG from other species. The binding factors were purified by concanavalin A affinity chromatography, an IgE immunoaffinity column and SDS-PAGE. This purification procedure resulted in five major proteins bands at 13, 15, 30, 60 and less than 150 kilodaltons. As judged by Western blot analysis, all bands were variably glycosylated and were capable of binding IgE. Biological activity resided even within the small molecular weight (13 kilodaltons) peptide in terms of inhibiting IgE synthesis of the U266 B cell line and net IgE synthesis of B cells from atopic blood donors. Additionally, semipurified binding factor preparations also inhibited the Pokeweed mitogen-induced IgG synthesis while stimulating IgE synthesis at the same time. At present it is not clear whether the different molecular entities are dimers or polymers of the same protein, or are all derived from one larger protein (proteolytically cleaved), or whether they are completely different peptides mediating similar immunoglobulin-binding capacities as well as similar biological activities. Glycosylation of binding factors is not responsible for binding to immunoglobulins, but might be important for its biological activity.
Antibody Formation↗
Effect of schistosome-derived inhibitory factor on the cell cycle of T lymphocytes.
A schistosome-derived inhibitory factor (SDIF) with immunosuppressive properties has been investigated for its effect on human T cell proliferation. We show here that SDIF has no effect on the process of lymphocyte activation because peripheral blood leukocytes (PBL) stimulated with lectin in the presence of SDIF increased normally their RNA content and showed normal acquisition of interleukin 2 (IL-2) and transferrin receptors. IL-2 production was not altered by SDIF but utilization of IL-2 was decreased, suggesting that SDIF blocked cells before or in the early s phase. Jurkat T cell line cells physically enriched for G1 cells were also more susceptible to SDIF inhibition. On the contrary, normal PBL or Jurkat cells which were already in the s phase were no more inhibited by SDIF. While SDIF has no effect on T lymphocyte activation and on production of regulatory lymphokines it selectively blocks T cell proliferation at G1 transition of the cell cycle.
Cell Cycle↗
[Current concepts in immunoregulation and its significance for allergies].
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Antibody Formation↗
Dot-immunobinding assay with monoclonal anti-IgE antibodies for the detection and quantitation of human IgE.
This paper describes a dot immunobinding assay for determining total human IgE with a tandem of monoclonal anti-IgE antibodies. Minute quantities of monoclonal anti-IgE antibodies were adsorbed on nitrocellulose discs. IgE bound to this solid phase monoclonal anti-IgE antibody was detected by a second monoclonal antibody conjugated with horseradish peroxidase. Using 4-chloro-1-naphthol as a chromogen results in a stable colour reaction that can be semiquantitatively analysed by the naked eye. The colour intensities of the reaction were also analysed by densitometry, yielding a very reproducible quantitation of human serum IgE. Using a serum dilution of 1:50, IgE could be detected in the range of 12.5-2500 U/ml. Using non-diluted serum samples IgE levels between 0.05-50 U/ml were reproducibly measured. Total serum IgE as determined by this dot assay correlated very well with IgE determinations performed by the commercial PRIST assay.
Antibodies, Monoclonal↗
Interference of a C3-fragment preparation with IL 2-dependent proliferation.
A C3-fragment preparation (C3-FP) was studied for its ability to regulate human peripheral blood lymphocyte activation. It was found that very low concentrations of this low m.w. fraction, which was free of C3a, inhibited the PHA-induced lymphocyte proliferation without any cytotoxicity. Cytofluorometric analysis showed that C3-FP did not influence the transition of T cells from the G0 to the G1a phase of the cell cycle. However, the IL 2-dependent transition from the G1a to the G1b phase of the cell cycle was effectively blocked. Addition of exogenous IL 2 did not release cells arrested in the G1a phase. Furthermore, neither IL 2 production nor IL 2 receptor formation was inhibited by C3-FP, and binding of IL 2 to its receptor was unaltered. It was found that only IL 2-dependent cell lines were inhibited in their proliferation; all other tested cell lines were unaffected by C3-FP. Our findings suggest that cleaved products of C3 may inhibit IL 2-dependent lymphocyte proliferation at a stage where the IL 2 signal is required for initiation of proliferation.
Complement C3↗