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h). For this purpose, a four-color multiple-staining direct immunofluorescence technique analyzed by flow cytometry was systematically used in all experiments (n ‫؍‬ 19). Our results show that after stimulation, an important proportion of each of the two CD33؉ myeloid DC subsets as well as the monocytes produce significant amounts of all cytokines analyzed under each of the experimental conditions assayed. In contrast, CD33؊/؉lo lymphoplasmocytoid DC failed to produce detectable levels of any of the above-mentioned cytokines under the same stimulatory conditions. Upon comparing the different stimuli used, LPS was associated with higher percentages of cytokine-producing cells compared with SAC, especially within the CD33 hi DC subset; interestingly, the addition of IFN-␥ enhanced the response of monocytes to both LPS and SAC. As regards the secretion-blocking agents, brefeldin A alone was superior to the combination of brefeldin A and monensin. This is because it was frequently associated with both a higher percentage of cytokine-positive cells and greater amounts of detectable cytokines per cell. Sequential analysis of cytokine production by PB DC and monocytes after 6, 12, and 24 h of cell culture showed that after 6 h, an increased cell death rate existed among DC, which became even undetectable at 24 h, in the absence of a significant increase in cytokine secretion. In summary, our results show that from the experimental conditions assayed in this paper, to induce cytokine production by normal human DC and monocytes, maximum response is obtained once PB samples are stimulated for 6 h with LPS (with or without IFN-␥) in the presence of brefeldin A alone. Cytometry (Comm. Clin. Cytometry) 46:33-40, 2001.

Background: Basophil activation tests (BATs) have been demonstrated to be useful in detecting IgEmediated sensitization by measuring basophil activation surface markers (CD63 and CD203c). Hymenoptera venom is one of the best known mediators-release trigger in patients with systemic mastocytosis (SM). The aim of this study was to investigate the use of BATs as an additional diagnostic tool in patients with mastocytosis suffering from hymenoptera venom anaphylaxis (HVA).Methods: A total of 22 patients with history of HVA and SM, together with a group of 11 patients with HVA in whom SM was ruled out after a complete bone marrow study, were analyzed.Results: Among 11 SM patients who had specific serum IgE (sIgE) against hymenoptera venom and an evaluable BAT, a positive BAT was found in nine. Additionally, a positive BAT was detected in three of seven patients who had no sIgE. These three patients had low levels of total IgE compared with control population (mean of 20 vs. 78 IU/mL); one had discontinued immunotherapy after 5 years, when sIgE levels had turned negative, and, in the other two patients, BAT identified the culprit insect.Conclusions: BAT is a useful complementary diagnostic tool to sIgE in mastocytosis patients with HVA, and it may contribute to predict or confirm these nearly fatal reactions, especially before discontinuing venom immunotherapy in patients who are negative for skin tests or sIgE or display low total IgE levels; in such cases, it also provides evidence on the culprit insect prompting HVA.

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Systemic mastocytosis (SM) is typically associated with CD25-positive mast cells (MCs) in the bone marrow (BM). The expression of CD25 in cutaneous MCs has shown to predict SM in adults. Recently, CD30 has emerged as a novel phenotypical marker of neoplastic MCs. The aim of this study was to investigate the patterns of expression of CD25 and CD30 on skin MCs in children with mastocytosis in the skin (MIS).Skin biopsies from 60 children with MIS were stained for H&E, c-kit, tryptase, CD25 and CD30. Intensity of expression and percentage of immunoreacting cells for the two latter markers were determined following a modified quickscore (Q-score) method. The potential association of clinical and laboratory features with CD25 Q-score and CD30 Q-score values was investigated.Overall, CD25 was expressed by cutaneous MCs in 28% of cases while CD30 in 82%. Positive CD25 expression was significantly more frequent in nodular forms vs. other subtypes of MIS but no correlations were found between CD25 Q-score values and gender, age at onset, extent of MIS, histological pattern, tryptase levels or time to diagnosis. By contrast, a multivariate ordinal regression analysis showed that CD30 Q-score negatively correlated with time elapsed from disease onset to diagnosis.Our results provide the first evidence that most children with MIS show an aberrant immunophenotype in their skin MCs. Based on previous studies in adults, it could be hypothesized that CD25 expression on skin MCs at pediatric ages might be associated with an increased risk for SM.