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Diagnosis of acute basophilic leukemia.

De novo acute basophilic leukemia (ABL) is a rare form of acute leukemia. Most frequently, the blast cells are morphologically undifferentiated, and the recognition of the presence of coarse basophilic granules may be the first step in diagnosis of this rare disorder. These granules are metachromatic and MPO negative. Immunophenotyping shows myeloid markers and some more specifically associated antigens such as CD9 or CD25 which are strongly expressed. Lymphoid, erythroid or megakaryocytic markers are not significantly expressed. In the absence of basophilic granules, some cases are classified as AML M0 if they express myeloid markers, or undifferentiated leukemia if no markers are present. If specific immature basophilic or theta granules are present, only an electron microscopic study will enable the diagnosis of a basophilic lineage assignment. Some cases may be misdiagnosed if all these steps are not followed. After all these investigations, two types of ABL may be defined: 1-A pure ABL, monophenotypic with basophilic lineage involvement alone, which should be classified as AML M8 . Genetic studies in these cases are very important for understanding the leukemic process and in a few cases, we can suspect c-MYB oncogene involvement but further investigations are still necessary. 2- More frequently, acute leukemia can be a mixture of blasts from different lineages with an important but variable participation of mature or immature basophilic cells. These cases must be classified as AML/Baso or multiphenotypic acute leukemias and often present Phl -chromosomal abnormality.

Biomarkers↗

Multiple defects in Fc epsilon RI signaling in Syk-deficient nonreleaser basophils and IL-3-induced recovery of Syk expression and secretion.

Human basophils respond to Ag-induced cross-linking of their high affinity IgE receptor, FcepsilonRI, by releasing histamine and other mediators from granules, producing IL-4 and other cytokines and, as shown in this study, by forming membrane ruffles and showing increased very late Ag-4 (VLA-4)-mediated adhesion to VCAM-1-expressing target cells. We have identified five blood donors whose basophils lack detectable levels of the FcepsilonRI-associated protein tyrosine kinase, Syk. Despite showing no obvious ultrastructural differences from normal basophils, nonreleaser basophils fail to form membrane ruffles, to show increased VLA-4-mediated adhesive activity, or to produce IL-4 in response to FcepsilonRI cross-linking. Although Syk protein levels are suppressed in basophils from all five donors, Syk mRNA is consistently present. Furthermore, culturing nonreleaser basophils for 4 days with IL-3 restores Syk protein expression and FcepsilonRI-mediated histamine release. Understanding the reversible suppression of Syk protein expression in nonreleaser basophils, and learning to replicate this property in patients with allergic inflammation could be a powerful and specific way to limit symptomatic disease.

Antibodies, Anti-Idiotypic↗

Evidence for the involvement of IgE-basophil system in acute serum sickness.

The role of the basophils in acute serum sickness of rabbits was examined by monitoring daily the absolute number of basophils before, during and after the disease period. After antigen (bovine serum albumin, BSA) elimination, levels of serum IgE and in vitro basophil degranulation in the presence of BSA were determined. The results showed that the onset of glomerular lesions depends upon the simultaneous occurrence of circulating immune complexes greater than 19 S and of an in vivo basophil depletion--probably equivalent to degranulation--reaching 70% of the pre-disease number. Post-disease antigen-dependent in vitro degranulation of the basophils and levels of serum IgE anti BSA did not prove to be good indexes of basophil sensitization. Our data suggest that basophils are instrumental at early stages of the deposition of immune complexes, most probably through their sensitization by membrane-bound IgE antibodies.

Animals↗

Eotaxin potentiates antigen-dependent basophil IL-4 production.

Basophils are a major source of IL-4, which is a critical factor in the generation of allergic inflammation. Eotaxin induces chemotaxis mediated through the CC chemokine receptor 3 (CCR3) present on basophils as well as eosinophils and Th2 cells, thereby promoting cell recruitment. To determine whether eotaxin has other proinflammatory activity, we examined the effect of eotaxin on basophil IL-4 expression by flow cytometry. Eotaxin alone had no effect on basophil IL-4 production, but further increased allergen-stimulated IL-4 expression. Eotaxin also enhanced IL-4 release from purified basophils 2- to 4-fold, as determined by ELISA (p < 0.01). Addition of eotaxin to cultures resulted in a 40-fold left shift in the dose response to Ag. This effect was obtained with physiologic concentrations of eotaxin (10 ng/ml), was abrogated by an Ab to the CCR3 receptor, and was noted with other chemokine ligands of CCR3. Additionally, eotaxin augmented IL-3 priming of basophil IL-4 production in a synergistic manner (p < 0.01). In contrast, no priming was observed with either IL-5 or GM-CSF. These results establish a novel function for eotaxin and other chemokine ligands of CCR3: the potentiation of Ag-mediated IL-4 production in basophils, and suggest a potential nonchemotactic role for CC chemokines in the pathogenesis and amplification of inflammation.

Adjuvants, Immunologic↗

IL-4 induces adherence of human eosinophils and basophils but not neutrophils to endothelium. Association with expression of VCAM-1.

The present studies were performed to explore potentially selective mechanisms of leukocyte adhesion in an attempt to understand how preferential recruitment of eosinophils and basophils might occur during allergic and other inflammatory reactions. Stimulation of human vascular endothelial cells for 24 h with IL-4 (30 to 1,000 U/ml) induced adhesion for eosinophils (up to approximately four-fold of control) and basophils (up to approximately twofold of control) but not neutrophils (less than 125% of control). Analysis of endothelial expression of adhesion molecules by flow cytometry revealed that IL-4 treatment induced vascular cell adhesion molecule-1 (VCAM-1) expression without significantly affecting the expression of other adhesion molecules, namely endothelial-leukocyte adhesion molecule-1 (ELAM-1) or intercellular adhesion molecule-1 (ICAM-1). The concentration-response curve for IL-4-induced VCAM-1 expression paralleled that for adhesion. Endothelial cells stimulated with IL-4 expressed adhesive properties for eosinophils by 3 h; the response increased steadily during a 24-h time course study. Eosinophils and basophils adhered to plates coated with a recombinant form of VCAM-1. This adhesion was blocked with antibodies to VCAM-1 but not ELAM-1. mAb directed against either VCAM-1 or VLA-4 inhibited (by approximately 75%) the binding of eosinophils and basophils to IL-4-stimulated endothelial cells. Because VLA-4 and VCAM-1 have been demonstrated to bind to each other in other adhesion systems, these results suggest that IL-4 stimulates eosinophil and basophil adhesion by inducing endothelial cell expression of VCAM-1 which binds to eosinophil and basophil VLA-4. The lack of expression of VLA-4 on neutrophils and the failure of IL-4 to stimulate neutrophil adherence support this conclusion. It is proposed that local release of IL-4 in vivo in allergic diseases or after experimental allergen challenge may partly explain the enrichment of eosinophils and basophils (vs neutrophils) observed in these situations.

Antibodies, Monoclonal↗

Anti-human IgG causes basophil histamine release by acting on IgG-IgE complexes bound to IgE receptors.

We have reexamined the ability of anti-human IgG antibodies to induce histamine release from human basophils. A panel of purified murine mAbs with International Union of Immunological Societies-documented specificity for each of the four subclasses of human IgG was used. Of the 24 allergic subjects studied, the basophils of 75% (18/24) released greater than 10% histamine to one or more anti-IgG1-4 mAb, whereas none of the 13 nonatopic donor's basophils released histamine after stimulation with optimal amounts of anti-IgG mAb. The basophils of 85% (11/13) of the nonatopic donors did respond to anti-IgE challenge, as did 92% (22/24) of the atopic donor cells. Histamine release was induced most frequently by anti-IgG3, and 10/18 anti-IgG responder cells released histamine with mAb specific for two or more different subclass specificities. The rank order for induction of histamine release was anti-IgG3 greater than anti-IgG2 greater than IgG1 greater than anti-IgG4. As in our previous study using polyclonal anti-IgG, 100- to 300-micrograms/ml quantities of the anti-IgG mAb were required for maximal histamine release, about 1000-fold higher than those for comparable release with anti-human IgE. Specificity studies using both immunoassays and inhibition studies with IgE myeloma protein indicated that anti-IgG induced histamine release was not caused by cross-reactivity with IgE. Ig receptors were opened by lactic acid treatment so that the cells could be passively sensitized. Neither IgE myeloma nor IgG myeloma (up to 15 mg/ml) proteins could restore the response to anti-IgG mAb. However, sera from individuals with leukocytes that released histamine upon challenge with anti-IgG mAb could passively sensitize acid-treated leukocytes from both anti-IgG responder and nonresponder donors for an anti-IgG response. The only anti-IgG mAb that induced release from these passively sensitized cells were those to which the serum donor was responsive. Sera from non-IgG responders could not restore an anti-IgG response. These data led to the hypothesis that the IgG specific mAb were binding to IgG-IgE complexes that were attached to the basophil through IgE bound to the IgE receptor. This was shown to be correct because passive sensitization to anti-IgG could be blocked by previous exposure of the basophils to IgE. We conclude that anti-IgG-induced release occurs as a result of binding to IgG anti-IgE antibodies and cross-linking of the IgE receptors on basophils.

Antibodies, Anti-Idiotypic↗

Type beta transforming growth factors promote interleukin-3 (IL-3)-dependent differentiation of human basophils but inhibit IL-3-dependent differentiation of human eosinophils.

Basophils and eosinophils share a common differentiation pathway. Factors regulating terminal commitment toward one cell type, however, have so far not been defined. Interleukin-3 (IL-3) is a potent differentiation factor for both human eosinophils and basophils. In the present study, the effects of various recombinant human (rh) growth regulators on IL-3-dependent growth of eosinophils and basophils were studied in a bone marrow (BM) suspension culture system (normal donors, n = 13). We found that type beta transforming growth factors (TGFs) lead to a significant increase in the absolute numbers of basophils in BM cultures grown in the presence of IL-3 (day 14 of culture; IL-3: 133 +/- 20 v IL-3 + TGF-beta 1: 231 +/- 28 x 10(3)/mL [P less than .01]) and to an increase in the total histamine values (IL-3: 72.6 +/- 22.2 v IL-3 + TGF-beta 1: 142.9 +/- 37.3 ng/mL [P less than .015]) compared with rhIL-3 alone. In contrast, type beta TGFs were found to inhibit the IL-3-dependent growth of eosinophils (IL-3: 170.4 +/- 37.2 v IL-3 + TGF-beta 1: 16.7 +/- 5.2 x 10(3)/mL [P less than .01]) and formation of eosinophil cationic protein in the same culture system. The effect of TGF-beta 1 (and TGF-beta 2) on IL-3-dependent differentiation of basophils and eosinophils was dose- and time-dependent (maximum effects observed with 1 to 10 ng/mL of rhTGF-beta 1 or TGF-beta 2) and could be neutralized by an antibody specific for TGF-beta 1. In contrast to the TGFs, interferon-alpha (IFN-alpha) and IFN-gamma were found to downregulate IL-3-dependent formation of both basophils (IL-3: 167 +/- 33 v IL-3 + IFN-alpha: 67 +/- 25 v IL-3 + IFN-gamma: 65 +/- 33 x 10(3)/mL [P less than .01]) and eosinophils (IL-3: 239 +/- 5 v IL-3 + IFN-alpha: 81 +/- 4 v IL-3 + IFN-gamma: 67 +/- 17 x 10(3)/mL [P less than .05]) in our culture system. Type beta TGFs as well as the IFNs failed to directly induce differentiation of human basophils or eosinophils in the absence of other growth factors. Together, these results show that type beta TGFs and IFNs are potent regulators of cytokine-dependent growth and differentiation of human allergic effector cells.

Antibodies, Monoclonal↗

Histamine release and morphological changes in basophilic granulocytes of atopic asthmatics induced by antigen, anti-IgE and Ca ionophore A 23187.

Morphological changes of basophils from atopic asthmatics were compared among antigen, anti-IgE and Ca ionophore A23187 stimulation. 1. Antigen induced rapid and marked increase of histamine release from basophils compared with Ca ionophore A23187. 2. The decrease in number of basophils following stimulation with the agents was significantly higher in antigen stimulation than in Ca ionophore A23187 stimulation. 3. The increased ratio of short to long axis diameter (L/Sb ratio) of the cells, which shows an increased motility of basophils, was significantly higher in antigen stimulation. The ratio did not change by stimulation with Ca ionophore A23187. 4. Stimulation of basophils by Ca ionophore A23187 induced marked increase in mean diameter (MD) of the cells. Activation of basophils by anti-IgE was similar to that by antigen, but slower in start and shorter in duration compared with antigen. The results show that an increase in motility is essential for release mechanism of chemical mediators from basophils in antigen and anti-IgE stimulation, but not in Ca ionophore A23187 stimulation.

Adult↗

Human basophils express interleukin-4 receptors.

Interleukin-4 (IL-4), a multipotential lymphokine reputed to play an important role in the regulation of immune responses, interacts with a variety of hemopoietic target cells through specific cell surface membrane receptors. The present study was designed to investigate whether human basophils express IL-4 binding sites. For this purpose, basophils were enriched to homogeneity (93% and 98% purity, respectively) from the peripheral blood of two chronic granulocytic leukemia (CGL) donors using a cocktail of monoclonal antibodies (MoAbs) and complement. Purified basophils bound 125I-radiolabeled recombinant human (rh) IL-4 in a specific manner. Quantitative binding studies and Scatchard plot analysis revealed the presence of a single class of high affinity IL-4 binding sites (280 +/- 40 sites per cell in donor 1 and 640 +/- 45 sites per cell in donor 2) with an apparent dissociation constant, kd, of 7.12 x 10(-11) +/- 2.29 x 10(-11) and 9.55 +/- 3.5 x 10(-11) mol/L, respectively. KU812-F, a human basophil precursor cell line, was found to express a single class of 810 to 1,500 high affinity IL-4 binding sites with a kd of 2.63 to 5.54 x 10(-10) mol/L. No change in the numbers or binding constants of IL-4 receptors was found after exposure of KU812-F cells to rhIL-3 (a potent activator of basophils) for 60 minutes. No effect of rhIL-4 on 3H-thymidine uptake, release or synthesis of histamine, or expression of basophil differentiation antigens (Bsp-1, CD11b, CD25, CD40, CD54) on primary human CGL basophils or KU812-F cells was observed.

Antigens, Surface↗

F-met peptide-induced degranulation of human basophils.

We examined the kinetics of morphologic change induced by stimulation of human basophils with f-Met peptide using partially purified cells from normal donors. Supernatants were collected at 30 and 60 minutes and assayed for histamine with an automated fluorometric technique. Samples of basophils were prepared for electron microscopy at 0, 10, 20, 30 seconds and 1, 2, 5 and 10 minutes poststimulation with f-Met peptide. We found that f-Met peptide, a bacterial peptide, induced a unique sequence of morphologic events that included morphologies we have previously identified and termed piecemeal degranulation in human basophils in situ as well as those induced by IgE mechanisms ex vivo and termed anaphylactic degranulation, thus supporting a general degranulation model for basophils and mast cells (Dvorak HF, Dvorak AM: In Clinics of Haematology, Granulocyte and Monocyte Abnormalities, Vol 4, edited by Lichtman MA, p 651. London, WB Saunders Co, Ltd, 1975). In addition to this degranulation continuum, we found that chambers of releasing granules underwent extraordinary increases in size as they emptied their contents and before their resolution by extrusion. The enlarging granule chambers accumulated numerous concentric dense membranes, vesicles, and Charcot-Leyden crystals. These early changes generally preceded 1/2-maximum histamine release, whereas the later extrusion of full granules, emptied granules and their membranous contents coincided with 1/2-maximum histamine release (Warner JA, Peters SP, Lichtenstein LM, Hubbard W, Yancey KB, Stevenson HC, Miller PJ, MacGlashan DW Jr. J Leukocyte Biol 45:558, 1989). Shedding of membranes from several sources accompanied extrusion of granules and intragranular Charcot-Leyden crystals. These sources included the expanded granule membranes from empty granules, granule membranes from full granules, collections of intragranular concentric dense membranes and vesicles, and surface membranes and processes. These extraordinary membrane shifts were generated and persisted over the 10-minute period examined and coincided with the later time frame within which leukotriene C4 was generated and released from human basophils stimulated by f-Met peptide (Warner JA, Peters SP, Lichtenstein LM, Hubbard W, Yancey KB, Stevenson HC, Miller PJ, MacGlashan DW Jr: J Leukocyte Biol 45:558, 1989). Viable basophils, completely free of both full and empty granules, showed some morphologic evidence of recovery of granule products by 10 minutes after stimulation with f-Met peptide. The unique morphology of f-Met peptide-induced degranulation of human basophils is supported by the uniqueness of the biochemical events associated with this trigger.(ABSTRACT TRUNCATED AT 400 WORDS)

Basophils↗

Oscillations in free cytosolic calcium during IgE-mediated stimulation distinguish human basophils from human mast cells.

Free cytosolic calcium ([Ca2+]i) levels increase after the stimulation of either human basophils or mast cells with anti-IgE antibody. Previous studies found that the mast cell [Ca2+]i response was graded in magnitude, according to stimulus strength, and that the activated level was free of oscillations. The current studies demonstrate several new features of the mast cell and basophil response. First, in mast cells, the transition to activated [Ca2+]i levels was abrupt (width = 6.5 +/- 2 s), after a period of quiescence whose duration (5 to 300 s) was a function of the strength of the stimulus. At optimal concentrations of anti-IgE, 97% of mast cells showed only abrupt transitions and oscillation-free activated calcium levels. In contrast, basophils showed marked oscillations whose magnitudes were partially dependent on the strength of stimulus. Like the mast cell, there was a quiescent period before the first transition and this period was also dependent on the strength of the stimulus. Oscillations were generally superimposed on an elevated [Ca2+]i level at a frequency of 0.5 to 3/min, had half-widths of 5 to 20 s, and were markedly chaotic in the frequency domain. In general, oscillations were more apparent at suboptimal concentrations of anti-IgE. Despite the apparent contrast in basophil and mast cell responses, oscillations (1 or 2 in a 10-min period) could be observed in a small percentage of mast cells and some basophils showed characteristics of mast cells. We tentatively conclude that mast cells and basophils utilize a similar mechanism of calcium mobilization but that the nonlinear characteristics of the calcium response may account for the mast cell/basophil differences. These studies indicated that the calcium kinetics, as measured by population averages, did not reflect the kinetics observed at the single cell level. Both mast cells and basophils had characteristics which could be described as graded and characteristics resembling all-or-nothing processes; the magnitude of a response was graded according the strength of stimulus while the kinetics profile appeared as an all-or-nothing event.

Antibodies, Anti-Idiotypic↗

Binding constants of IgE receptors on human blood basophils for IgE.

The ability to remove receptor-bound IgE but maintain functional integrity of human blood basophils allowed measurement of several parameters of binding of IgE. Non-specific binding was determined by a modification of the standard technique and was found to be only about 10% of specific binding. The number of free receptors varied inversely with the serum IgE of the donor. Forward rate constants were readily measured at 0 degree but were considerably higher at 37 degrees, and a rough estimate of the activation energy for binding was calculated to be 6700 calories/mol. Saturation of basophil receptors with IgE occurred in 1-3 h using 3.6 micrograms 125I-IgE/ml at 37 degrees. For seven donors, the forward rate constant, k1, ranged from 2.9 to 7.25 X 10(4) M/sec with a mean of 5.2 X 10(4)/M/sec. This is about one-third the reported value for cultured cord blood basophils. The backward rate constant, k-1, ranged from 1.4 to 3.9 X 10(-5)/sec with a mean of 2.5 X 10(-5)/sec. This is less than half that of cord blood basophils. The equilibrium association constant, Ka, ranged from 1.4 to 2.7 X 10(9)/M with a mean of 2.1 X 10(9)/M. This is comparable to the high affinity binding reported for cord blood and other basophils. There was no great difference in binding constants among the four atopic and three non-atopic donors studied. The affinity of monoclonal mouse 125I-IgE for IgE receptors on human basophils was about four-fold lower than that of human IgE. Sensitization of human basophils for histamine release with mouse IgE anti-DNP was confirmed. Non-specific human IgE but not IgG inhibited sensitization by mouse IgE, indicating that the same receptors were involved and that they were specific for IgE.

Basophils↗

Effects of a dihydropyridine calcium antagonist and agonist on human basophil histamine release.

Calcium influx is important to basophil histamine release, and even though a cromolyn-binding protein has been proposed to constitute a Ca2+ channel, the pathway of Ca2+ influx or involvement of a Ca2+ channel in this process has yet to be established. We evaluated the effects of a dihydropyridine antagonist, nitrendipine, and agonist, BAY k 8644, on human basophil histamine release. Nitrendipine inhibited ragweed antigen E-dependent basophil histamine release in a dose-dependent fashion with a 50% inhibitory dose of 3.7 (+/- 1.1) X 10(-6) mol/L, and maximal inhibition of histamine release (41.8% +/- 7.1%) was achieved with 1.0 X 10(-5) mol/L nitrendipine. Increased extracellular concentrations of Ca2+ reduced nitrendipine inhibition of histamine release. In contrast, the Ca2+ agonist, BAY k 8644, enhanced antigen E-dependent histamine release with an ED50 value of 5.0 (+/- 1.1) X 10(-6) mol/L. BAY k 8644 by itself, however, did not cause basophil histamine release nor did it enhance histamine release to the calcium ionophore A23187. Further, when the effects of BAY k 8644 on basophil histamine release were evaluated in the presence of nitrendipine, the enhancing action of BAY k 8644 was diminished in a competitive fashion. Therefore, even though these compounds act at specific Ca2+ channels in other tissues, our data do not establish either the presence of such channels in the IgE-dependent histamine release process of basophils or the mechanism of action for dihydropyridines in basophil histamine secretion.(ABSTRACT TRUNCATED AT 250 WORDS)

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗

Human monocytes generate basophil histamine-releasing activities.

Human peripheral blood monocytes generated activities during 24-h culture that were capable of triggering histamine release from 17 of 18 human basophil donors. Monocytes and their in vitro transformed macrophages continued to elaborate these basophil histamine-releasing activities for at least 3 wk in culture. In the 18 basophil donors tested, maximum histamine release induced by monocyte supernatants was 33.8 +/- 5.9% (mean +/- SEM) of total basophil histamine content; optimum anti-IgE-induced release was 38.8 +/- 6.2%. Basophil histamine release in response to monocyte activities was optimal at 37 degrees C and at calcium concentrations of 2 to 5 mM. Release was greater than 90% complete 1 min after challenge and was inhibited by anti-allergic drugs. The mechanism of release appeared to be independent of IgE binding. Gel filtration of supernatants derived from both day 1 (monocyte stage) and day 14 (macrophage stage) cultures demonstrated activity peaks with approximate m.w. of 12,000 and 30,000. In contrast to the marked responsiveness of basophils, only 2 of 10 human lung mast cell preparations responded; release in those preparations was low: 3% and 13% histamine release, respectively. Thus, monocytes produce potent histamine-releasing activities with differential actions on basophils and mast cells.

Basophils↗

Natural cytotoxic (NC) cell activity in basophilic cells: release of NC-specific cytotoxic factor by IgE receptor triggering.

A murine interleukin 3 (IL 3)-dependent basophilic mast cell line, PT-18 (A17), and a rat basophilic leukemic cell line, RBL-2H3, were shown to be capable of selective natural cytotoxic (NC) but not natural killer (NK) cell activity. The basophilic cell types could also be augmented in their NC activity by bridging of their surface IgE receptors. IgE-mediated triggering of the basophilic cells was accomplished by coating the cells with IgE and exposing the IgE-bound cells to specific antigen or to anti-IgE monoclonal antibody. Another method of triggering was by direct binding of basophilic cells to anti-IgE receptor monoclonal antibody. Basophilic cells, triggered by these methods, not only displayed increased NC activity but also released a soluble factor capable of selectively lysing NC tumor targets, WEHI-164, but not three of the NK-sensitive targets, YAC-1, RLM1, and RBL-5. Normal C3H/HeJ mouse embryonic fibroblasts were also not lysed. Dose response and time course of the cytotoxic factor release from triggered RBL-2H3 cells were similar to those of tritiated serotonin release. As with serotonin or histamine release, the NC-specific cytotoxic factor (NCCF) was not released in the absence of extracellular calcium. Therefore, NCCF appears to be released along with other mediators during the triggering of basophilic cells by bridging of IgE receptors. The m.w. of the native form of this factor, determined by a gel filtration method, was about 43,000.

Animals↗

[Inhibition of degranulation of human basophils by calcium antagonists].

The human basophil degranulation test (HBDT) quantifies the apparent disappearance of basophils after contact with the sensitizing allergen. The inhibition of degranulation by pharmacological products can be appreciated by incubating beforehand the basophils with the substance concerned during various times. The inhibitory effect is evaluated by the difference of degranulation with or without it. EDTA, sodium cromoglycate, verapamil and bepridil have been tested at different concentrations and at 3 times of basophils preincubation (0,15 and 30 minutes). Results obtained confirm previous reports concerning cromoglycate which didn't inhibit degranulation of basophils. EDTA at 3.4*10(-2)M and 3.4*10(-3)M concentration has an inhibitory effect, not increasing with the time of preincubation. The inhibition with verapamil is of the same order at 1*10(-4)M for the 3 times of incubation and weaker, but significant, at 1*10-M5 after 30 minutes of preincubation. Bepridil inhibits at 1*10(-4)M, but this effect disappears if basophils are preincubated 30 minutes with the drug. The inhibition of the specific basophil degranulation by these drugs is probably du to their calcium antagonist property. However their effect is not quite similar when cells are preincubated at different times with the drug. On the other hand, one can study with HBDT the inhibitory effect of a new product, even if the therapeutic indications would have to be studied afterwards.

Basophils↗

Detection of basophils growing in semisolid agar culture.

The production of basophils in semisolid agar cultures form normal and chronic myeloid leukemia (CML) committed granulocyte-macrophage precursors was investigated using an original whole-dish staining technique with toluidine blue which produces a specific metachromasia in basophils. As additional proof of the basophilic nature of metachromatic cells, their degranulation after challenge with C5a anaphylotoxin and Synachten was observed. Our studies show that few basophils are produced in cultures form normal bone marrow. CML CFUc produce more basophils, their number being roughly correlated with the degree of basophilia. We observed only clusters composed by a pure basophilic population, while larger aggregates in which basophils could be detected were composed also by other granulocytic cells.

Agar↗

Identification of pure and mixed basophil colonies in culture of human peripheral blood and marrow cells.

We present a colony assay system that allows in situ identification of human basophil/mast cell (basophil) colonies. In methylcellulose culture, in the presence of phytohemagglutinin-leukocyte conditioned media (PHA-LCM), human peripheral blood and bone marrow cells form colonies that can be distinguished by their unique morphological characteristics. Pure basophil colonies are diffuse, small colonies containing small, round, highly refractile cells. These characteristics of the constituent cells led us to the observation that a significant number of basophils are found in combination with eosinophils. The mixed eosinophil/basophil colonies have the distinctive elements of pure eosinophil and pure basophil colonies. Usually, these are diffuse colonies with compact clusters of slightly larger, darker-appearing cells. We also found colonies that contained basophils and neutrophils/monocytes, but this type could not be consistently identified by in situ morphology. Cytochemical analysis confirmed the metachromatic nature of the granules in the basophils. The presence of IgE receptors on the cells was documented by indirect immunofluorescent staining after passive sensitization with purified human IgE. Peripheral blood cells from six healthy volunteers formed 5.7 +/- 1.0 (mean +/- SEM) pure colonies in 2 X 10(5) cells. Cultures of bone marrow cells from patients with various types of anemia had 9.0 +/- 1.5 colonies in 10(5) cells. This is the first description of a colony assay system for in situ identification of a pure population of basophilic granulocytes.

Adult↗