Comparison of CD63 Upregulation Induced by NSAIDs on Basophils and Monocytes in Patients with NSAID Hypersensitivity

Background. An in vitro basophil activation test, based on the detection of CD63 upregulation induced by NSAIDs, has been described. Its clinical significance remains controversial. Objectives. In patients with a history of nonallergic NSAID hypersensitivity, stratified according to the severity of the symptoms, to assess with NSAIDs the predictive value of basophil (BAT) and monocyte (MAT) activation tests. Patients/Methods. Sixty patients who had NSAIDs-induced or exacerbated urticaria/angiooedema and 20 controls was included. After incubation with NSAIDs or acetaminophen, leukocytes were analysed for CD63 upregulation. Results. With aspirin, the sensitivity (37%) and specificity (90%) of BAT agree with already published results. In contrast, when patients had had cutaneous and visceral reactions, the frequency of positive BAT 14/22 (64%, P < 0.001) or MAT 10/22 (46%, P < 0.01) were increased. Conclusions. Positive tests were more frequent among patients having a severe hypersensitivity contrasting with the other patients who had results similar to controls

Influence of flanking sequence context on the conformational flexibility of aminofluorene-modified dG adduct in dA mismatch DNA duplexes

When positioned opposite to a dA in a DNA duplex, the prototype arylamine–DNA adduct [N-(2′-deoxyguanosin-yl)-7-fluoro-2-aminofluorene (FAF)] adopts the so-called ‘wedge’ (W) conformation, in which the carcinogen resides in the minor groove of the duplex. All 16 FAF-modified 12-mer NG*N/NAN dA mismatch duplexes (G* = FAF, N = G, A, C, T) exhibited strongly positive induced circular dichroism in the 290–360 nm range (ICD290–360 nm), which supports the W conformation. The ICD290–360 nm intensities were the greatest for duplexes with a 3′-flanking T. The AG*N duplex series showed little adduct-induced destabilization. An exception was the AG*T duplex, which displayed two well-resolved signals in the 19F NMR spectra. This was presumably due to a strong lesion-destabilizing effect of the 3′-T. The flanking T effect was substantiated further by findings with the TG*T duplex, which exhibited greater lesion flexibility and nucleotide excision repair recognition. Adduct conformational heterogeneity decreased in order of TG*T > AG*T > CG*T > AG*A > AG*G > AG*C. The dramatic flanking T effect on W-conformeric duplexes is consistent with the strong dependence of the ICD290-360 on both temperature and salt concentration and could be extended to the arylamine food mutagens that are biologically relevant in humans

Differential response of human basophil activation markers: a multi-parameter flow cytometry approach

<p>Abstract</p> <p>Background</p> <p>Basophils are circulating cells involved in hypersensitivity reactions and allergy but many aspects of their activation, including the sensitivity to external triggering factors and the molecular aspects of cell responses, are still to be focused. In this context, polychromatic flow cytometry (PFC) is a proper tool to investigate basophil function, as it allows to distinguish the expression of several membrane markers upon activation in multiple experimental conditions. </p> <p>Methods</p> <p>Cell suspensions were prepared from leukocyte buffy coat of K2-EDTA anticoagulated blood specimens; about 1500-2500 cellular events for each tested sample, gated in the lymphocyte CD45dim area and then electronically purified as HLADRnon expressing/CD123bright, were identified as basophilic cells. Basophil activation with fMLP, anti-IgE and calcium ionophore A23187 was evaluated by studying up-regulation of the indicated membrane markers with a two-laser six-color PFC protocol.</p> <p>Results</p> <p>Following stimulation, CD63, CD13, CD45 and the ectoenzyme CD203c up-regulated their membrane expression, while CD69 did not; CD63 expression occurred immediately (within 60 sec) but only in a minority of basophils, even at optimal agonist doses (in 33% and 14% of basophils, following fMLP and anti-IgE stimulation respectively). CD203c up-regulation occurred in the whole basophil population, even in CD63non expressing cells. Dose-dependence curves revealed CD203c as a more sensitive marker than CD63, in response to fMLP but not in response to anti-IgE and to calcium ionophore.</p> <p>Conclusion</p> <p>Use of polychromatic flow cytometry allowed efficient basophil electronic purification and identification of different behaviors of the major activation markers. The simultaneous use of two markers of activation and careful choice of activator are essential steps for reliable assessment of human basophil functions.</p