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On activation of human basophilic granulocytes with anti-IgE or with the chemotactic peptide, formyl-methionyl-leucyl-phenylalanine, the expression of the CD63 antigen on the cell surface, detected by monoclonal antibody (MAb) 435, increased up to 100-fold. The kinetics of CD63 up regulation and histamine release were identical, and a strong correlation was found between percentage of MAb 435-binding basophils and extent of histamine release. Immunoelectronmicroscopy demonstrated that the epitope for MAb 435 in resting basophils is located on the basophilic granule membrane. After basophil activation, MAb 435 bound to the exterior of the plasma membrane. Experiments with various doses of anti-IgE demonstrated that the binding of MAb 435 to basophilic granulocytes follows an all-or-nothing-like response per cell. Basophils either do not bind the MAb at all, or they bind a maximal amount of the MAb. We also measured the up regulation of the CD11CD18 leukocyte adhesion complex. Here, too, we noted an increase in cell-surface exposure of all subunits after activation. This increase was not as strong as increase found with MAb 435. Thus, MAb 435 is an interesting new tool for investigating the activation of human basophils, in addition to the measurement of mediator release. This MAb may be useful for the detection of basophil activation in vivo.

Background Better understanding of the relationship between antibody response to peanut and clinical sensitivity might lead to more accurate prognostication. Objective We sought to investigate peanut-specific IgE and IgG4 epitope diversity in relation to challenge-defined clinical sensitivity to peanut in a group of peanut-sensitized children. Methods Clinical sensitivity was determined by means of double-blind, placebo-controlled peanut challenges in 24 sensitized children. Six atopic control subjects were included. Specific IgE and IgG4 binding to 419 overlapping 15-amino-acid peptides representing the sequence of recombinant Ara h 1, Ara h 2, and Ara h3 was analyzed by means of microarray immunoassay. Results Peanut-sensitized patient sera bound significantly more IgE and IgG4 epitopes than control sera. This patient group reacted to the same Ara h 1, Ara h 2, and Ara h 3 epitopes as reported previously. There was a positive correlation between IgE epitope diversity (ie, number of epitopes recognized) and clinical sensitivity ( r = 0.6), such that patients with the greatest epitope diversity were significantly more sensitive than those with the lowest diversity ( P = .021). No specific epitopes were associated with severe reactions to peanut. IgG4 binding was observed to largely similar epitopes but was less pronounced than IgE binding and did not relate to the clinical sensitivity to peanut. IgE and IgG4 epitope-recognition patterns were largely stable over a 20-month period. Conclusion Clinical sensitivity, as determined by means of double-blind, placebo-controlled peanut challenge, is positively related to a more polyclonal IgE response, which remains stable over time.

Rationale IgE-binding peanut proteins smaller than 15 kDa were previously identified as potential allergens in the majority of a peanut allergic population. We characterized the novel allergen and determined whether it was similar to one of the thus far identified recombinant peanut allergens (Ara h1 -7). Methods The IgE-binding protein of Results The purified protein is a monomeric protein with a molecular weight of 14,981 Da as determined with MALDI-TOF mass-spectroscopy. The amino acid sequence of the first 39 N-terminal residues is identical to that of Ara h6, indicating that the allergen is Ara h6. It is recognized by 20 out of 30 peanut-allergic patients on IgE-immunoblot, and its potent biological functionality is demonstrated by the release of histamine from basophils, even at concentrationsbelow 10 pg/ml. Ara h6 has homology with Ara h2, especially in the mid part and the C-terminal part of the protein. Almost complete inhibition of IgE-Ara h6 interaction with Ara h2 demonstrates that at least part of the epitopes of Ara h6 is cross-reactive with epitopes on Ara h2. The partial inhibition of Ara h2-IgE interaction with Ara h6 indicates that next to shared epitopes, unique IgE-binding epitopes on Ara h2 exists. Conclusions Peanut-derived Ara h 6 is a biologically active allergen recognized by the majority of our patient population.

Meta-iodo-benzylguanidine (MIBG; 3 × 10 −5 M), a novel inhibitor of mono(ADP-ribosylation) -and the general ribosylation inhibitor nicotinamide (NA; 5–20 mM) both stimulated the glucocorticoid-mediated lysis of sensitive L1210 leukemia cells and even induced susceptibility in various human and murine lines refractory or resistant to dexamethasone (DEX). Potentiation and induction of DEX-sensitivity by ADP-ribosylation inhibitors was accompanied by an increase in saturable of 3 H-DEX binding sites and by a 2–3 fold increase in the affinity of intracellular receptors for hormone binding. Moreover, the ribosylation inhibitors converted the glucocorticoid antagonist RU-486 into a potent agonist for cytolysis of L1210 cells. We conclude that the cytolytic action of glucocorticoid hormones in leukemic cells is negatively controlled by (mono)ADP-ribosylation of receptor proteins.

Human monocytic cell fractions obtained by counterflow elutriation centrifugation (with regard to specific monocyte/macrophage characteristics: 82–88 % were positive for nonspecific esterase; 86–94 % for CD14) were cultured (overnight, 37°C) under nonadhering conditions (polypropylene tubes).Thirty to 40 % of the cells were found to differentiate into large, monocytoid cells with a dendritic morphology.These cells expressed a marker of active dendritic cells RFD1 in 76–89 % and were also positive for class II MHC antigens as identified by OKIa (95–97 %).An exposure of the blood monocytic cells to metrizamide (30 min, 14.5 %) prior to the overnight culture enhanced this differentiation, and 47–58 % of cells with a dendritic morphology were found (of which 80–87% RFD1 positive, and 95–97 Class II MHC positive). The cultured cell populations containing the cells with the morphology and marker pattern typifying dendritic cells, appeared functionally more active as accessory populations when compared to freshly isolated blood monocytic cell fractions; the cultured cells had an enhanced stimulator.capability in MLR, and cluster formation with lymphocytes was more prominent.At the same time, the cultured cell population showed a decreased bactericidal activity when compared to the freshly isolated monocytic populations, and in addition, all the cultured monocytoid cells had lost non-specific-esterase activity, while only approximately 10 % of cells were still positive for the CD14 marker. When U937 cells were exposed to metrizamide (14.5 % concentration, 30 min) and cultured under nonadhering conditions for 36 hours, similar changes were observed (30–45 became dendritic, 20 % RFD1 positive). Our data suggest that cells within the blood monocyte pool and the U937 cell line can differentiate into functionally active dendritic cells when maintained in culture under non-adhering conditions.An exposure to metrizamide prior to culture enhances this differentiation.

Ultraviolet B radiation can suppress cellular immunity. One of the mechanisms related to this immunosuppression is the disappearance of Langerhans cells from the epidermis. The aim of this study was to establish the mechanism of ultraviolet B-induced Langerhans cell disappearance in healthy individuals. The two most likely mechanisms for Langerhans cell disappearance are apoptosis and migration. Apoptosis was assessed in vivo by exposing buttock skin of 10 healthy volunteers to six minimal erythema doses of ultraviolet B. Only very few apoptotic Langerhans cells could be observed in sections from the ultraviolet B-exposed skin. Migration of Langerhans cells cannot be established in skin sections and suction blisters were therefore raised in an attempt to trap migrating Langerhans cells in the sub-basal membrane blister fluid. Blisters were raised on the flexor side of the lower arm of 30 healthy volunteers at several time points after exposure of the skin to six minimal erythema doses of ultraviolet B. Blister fluid was collected and blister roofs were removed to check for Langerhans cell disappearance. Langerhans cells were detected in the blister fluid of the ultraviolet B-exposed skin and not of the unexposed skin. The number of Langerhans cells in the blister fluid peaked at about 18 h after ultraviolet exposure, which coincided with the largest depletion of Langerhans cells in the blister roof. A fraction (20–30%) of the Langerhans cells in the blister fluid stained positive for DNA damage (cyclobutyl pyrimidine dimers), showing that they originated from the epidermis. Ultraviolet B-induced Langerhans cell disappearance appears to be mainly attributable to migration.

Atopic dermatitis (AD) is a complex skin disease frequently associated with other diseases of the atopic diathesis. Recent evidence supports the concept that AD can also recognize other comorbidities, such as chronic inflammatory bowel or cardiovascular diseases. These comorbidities might result from chronic cutaneous inflammation or from a common, yet-to-be-defined immunologic background leading to immune deviations. The activation of immune cells and their migration to the skin play an essential role in the pathogenesis of AD. In patients with AD, an underlying immune deviation might result in higher susceptibility of the skin to environmental factors. There is a high unmet medical need to define immunologic endotypes of AD because it has significant implications on upcoming stratification of the phenotype of AD and the resulting targeted therapies in the development of precision medicine. This review article emphasizes studies on environmental factors affecting AD development and novel biological agents used in the treatment of AD. Best evidence of the clinical efficacy of novel immunologic approaches using biological agents in patients with AD is available for the anti-IL-4 receptor α-chain antibody dupilumab, but a number of studies are currently ongoing with other specific antagonists to immune system players. These targeted molecules can be expressed on or drive the cellular players infiltrating the skin (eg, T lymphocytes, dendritic cells, or eosinophils). Such approaches can have immunomodulatory and thereby beneficial clinical effects on the overall skin condition, as well as on the underlying immune deviation that might play a role in comorbidities. An effect of these immunologic treatments on pruritus and the disturbed microbiome in patients with AD has other potential consequences for treatment.

To understand the hematopoietic and nonhematopoietic responses to interleukin-3 (IL-3), expression of cell-surface IL-3 receptors (IL-3R) was examined on bone marrow (BM) cells and peripheral blood (PB) cells of rhesus monkeys during the course of in vivo IL-3 treatment. Whereas IL-3R expression is low in untreated monkeys, IL-3 administration led to a gradual increase in both low- and high-affinity binding sites for IL-3. This increase reflected the total number of cells expressing IL- 3Rs, as detected by flow cytometry using biotinylated IL-3. Most of these IL-3R+ cells in both BM and PB could be characterized as basophilic granulocytes that contained high levels of histamine. In contrast to the effect on these differentiated cells, IL-3 administration did not significantly alter the low level IL-3R expression on immature, CD34+ cells. Further flow cytometric analysis using biotinylated growth factors showed that the IL-3R+ basophils also expressed receptors for granulocyte-macrophage colony-stimulating factor (GM-CSF), but not for IL-6 or Kit ligand. These findings indicated that the IL-3R+ cells included neither monocytes, which express GM-CSFRs and IL-6Rs abundantly, nor mast cells, which express c- kit. By combining flow cytometric and Scatchard data, it was calculated that the basophils contain as many as 1 to 2 x 10(3) high-affinity IL- 3Rs and 15 to 30 x 10(3) low-affinity sites. The finding that in vivo IL-3 treatment leads to the production of large numbers of cells that express high levels of IL-3R and are capable of producing histamine provides an explanation for the often severe allergic reactions that occur during prolonged IL-3 administration. It also indicates that IL- 3, in addition to its direct effects on hematopoietic cells, may also stimulate hematopoiesis through the release of secondary mediators such as histamine by IL-3-responsive mature cells.