T cell-mediated hypersensitivity to quinolones: mechanisms and cross-reactivity

BACKGROUND: Quinolones are widely used, broad spectrum antibiotics that can induce immediate- and delayed-type hypersensitivity reactions, presumably either IgE or T cell mediated, in about 2-3% of treated patients. OBJECTIVE: To better understand how T cells interact with quinolones, we analysed six patients with delayed hypersensitivity reactions to ciprofloxacin (CPFX), norfloxacin (NRFX) or moxifloxacin (MXFX). METHODS: We confirmed the involvement of T cells in vivo by patch test and in vitro by means of the lymphocyte proliferation test (LTT). The nature of the drug-T cell interaction as well as the cross-reactivity with other quinolones were investigated through the generation and analysis (flow cytometry and proliferation assays) of quinolone-specific T cell clones (TCC). RESULTS: The LTT confirmed the involvement of T cells because peripheral blood mononuclear cells (PBMC) mounted an enhanced in vitro proliferative response to CPFX and/or NRFX or MXFX in all patients. Patch tests were positive after 24 and 48 h in three out of the six patients. From two patients, CPFX- and MXFX-specific CD4(+)/CD8(+) T cell receptor (TCR) alphabeta(+) TCC were generated to investigate the nature of the drug-T cell interaction as well as the cross-reactivity with other quinolones. The use of eight different quinolones as antigens (Ag) revealed three patterns of cross-reactivity: clones exclusively reacting with the eliciting drug, clones with a limited cross-reactivity and clones showing a broad cross-reactivity. The TCC recognized quinolones directly without need of processing and without covalent association with the major histocompatability complex (MHC)-peptide complex, as glutaraldehyde-fixed Ag-presenting cells (APC) could present the drug and washing quinolone-pulsed APC removed the drug, abrogating the reactivity of quinolone-specific TCC. CONCLUSION: Our data show that T cells are involved in delayed immune reactions to quinolones and that cross-reactivity among the different quinolones is frequent.

New ways of looking at synapses

Current concepts of synaptic fine-structure are derived from electron microscopic studies of tissue fixed by chemical fixation using aldehydes. However, chemical fixation with glutaraldehyde and paraformaldehyde and subsequent dehydration in ethanol result in uncontrolled tissue shrinkage. While electron microscopy allows for the unequivocal identification of synaptic contacts, it cannot be used for real-time analysis of structural changes at synapses. For the latter purpose advanced fluorescence microscopy techniques are to be applied which, however, do not allow for the identification of synaptic contacts. Here, two approaches are described that may overcome, at least in part, some of these drawbacks in the study of synapses. By focusing on a characteristic, easily identifiable synapse, the mossy fiber synapse in the hippocampus, we first describe high-pressure freezing of fresh tissue as a method that may be applied to study subtle changes in synaptic ultrastructure associated with functional synaptic plasticity. Next, we propose to label presynaptic mossy fiber terminals and postsynaptic complex spines on CA3 pyramidal neurons by different fluorescent dyes to allow for the real-time monitoring of these synapses in living tissue over extended periods of time. We expect these approaches to lead to new insights into the structure and function of central synapses.

Lectin binding patterns in two cultured endothelial cell types derived from bovine corpus luteum

Epithelial cells of different phenotypes derived from bovine corpus luteum have been studied intensively in our laboratory. In this study, specific lectin binding was examined for cells of type 1 and 3, which were defined as endothelial cells. In order to confirm differences in their glycocalyx at the light microscopic level, five biotinylated lectins were applied to postconfluent cultures which had been fixed with buffered paraformaldehyde or glutaraldehyde. Cells were not permeabilized with any detergent. Lectin binding was localized with a streptavidin-peroxidase complex which was visualized with two different techniques. The DAB technique detected peroxidase histochemically, while the immunogold technique used an anti-peroxidase gold complex together with silver amplification. Neither cell type 1 nor cell type 3 bound a particular lectin selectively, yet each cell type expressed a particular lectin binding pattern. With the DAB technique, diverse lectin binding patterns were seen, probably indicating either "outside" binding, i.e., a diffuse pattern, a lateral-cell-side pattern and a microvillus-like pattern, or "inside" binding, i.e., a diffuse pattern, and a granule-like pattern. With the immunogold technique, only "outside" binding was observed. In addition, the patterns of single cilia or of single circles were detected, the latter roughly representing 3-micron-sized binding sites for concanavalin A. When localizing them at the ultrastructural level, single circles corresponded with micron-sized discontinuities of the plasma membrane. Shedding vesicles were detected whose outer membrane was labelled with concanavalin A. Our results confirm the diversity of the two cell types under study. The "inside" lectin binding may be caused by way of transient plasma membrane openings and related to shedding of right-side out vesicles ("ectocytosis").