Direct binding of peptides to MHC class I molecules on living cells. Analysis at the single cell level.

To directly assess the binding of exogenous peptides to cell surface-associated MHC class I molecules at the single cell level, we examined the possibility of combining the use of biotinylated peptide derivatives with an immunofluorescence detection system based on flow cytometry. Various biotinylated derivatives of the adenovirus 5 early region 1A peptide 234-243, an antigenic peptide recognized by CTL in the context of H-2Db, were first screened in functional assays for their ability to bind efficiently to Db molecules on living cells. Suitable peptide derivatives were then tested for their ability to generate positive fluorescence signals upon addition of phycoerythrin-labeled streptavidin to peptide derivative-bearing cells. Strong fluorescent staining of Db-expressing cells was achieved after incubation with a peptide derivative containing a biotin group at the C-terminus. Competition experiments using the unmodified parental peptide as well as unrelated peptides known to bind to Kd, Kb, or Db, respectively, established that binding of the biotinylated peptide to living cells was Db-specific. By using Con A blasts derived from different H-2 congenic mouse strains, it could be shown that the biotinylated peptide bound only to Db among > 20 class I alleles tested. Moreover, binding of the biotinylated peptide to cells expressing the Dbm13 and Dbm14 mutant molecules was drastically reduced compared to Db. Binding of the biotinylated peptide to freshly isolated Db+ cells was readily detectable, allowing direct assessment of the relative amount of peptide bound to distinct lymphocyte subpopulations by three-color flow cytometry. While minor differences between peripheral T and B cells could be documented, thymocytes were found to differ widely in their peptide binding activity. In all cases, these differences correlated positively with the differential expression of Db at the cell surface. Finally, kinetic studies at different temperatures strongly suggested that the biotinylated peptide first associated with Db molecules available constitutively at the cell surface and then with newly arrived Db molecules.

Superficial and deep changes of cellular mechanical properties following cytoskeleton disassembly.

The cytoskeleton, composed of actin filaments, intermediate filaments, and microtubules, is a highly dynamic supramolecular network actively involved in many essential biological mechanisms such as cellular structure, transport, movements, differentiation, and signaling. As a first step to characterize the biophysical changes associated with cytoskeleton functions, we have developed finite elements models of the organization of the cell that has allowed us to interpret atomic force microscopy (AFM) data at a higher resolution than that in previous work. Thus, by assuming that living cells behave mechanically as multilayered structures, we have been able to identify superficial and deep effects that could be related to actin and microtubule disassembly, respectively. In Cos-7 cells, actin destabilization with Cytochalasin D induced a decrease of the visco-elasticity close to the membrane surface, while destabilizing microtubules with Nocodazole produced a stiffness decrease only in deeper parts of the cell. In both cases, these effects were reversible. Cell softening was measurable with AFM at concentrations of the destabilizing agents that did not induce detectable effects on the cytoskeleton network when viewing the cells with fluorescent confocal microscopy. All experimental results could be simulated by our models. This technology opens the door to the study of the biophysical properties of signaling domains extending from the cell surface to deeper parts of the cell.

A novel cyclosporin a aqueous formulation for dry eye treatment: in vitro and in vivo evaluation.

PURPOSE: The aim of the present study was the in vitro and in vivo evaluation of a novel aqueous formulation based on polymeric micelles for the topical delivery of cyclosporine A for dry eye treatment. METHODS: In vitro experiments were carried out on primary rabbit corneal cells, which were characterized by immunocytochemistry using fluorescein-labeled lectin I/isolectin B4 for the endothelial cells and mouse monoclonal antibody to cytokeratin 3+12 for the epithelial ones. Living cells were incubated for 1 hour or 24 hours with a fluorescently labeled micelle formulation and analyzed by fluorescence microscopy. In vivo evaluations were done by Schirmer test, osmolarity measurement, CyA kinetics in tears, and CyA ocular distribution after topical instillation. A 0.05% CyA micelle formulation was compared to a marketed emulsion (Restasis). RESULTS: The in vitro experiments showed the internalization of micelles in the living cells. The Schirmer test and osmolarity measurements demonstrated that micelles did not alter the ocular surface properties. The evaluation of the tear fluid gave similar CyA kinetics values: AUC = 2339 ± 1032 min*μg/mL and 2321 ± 881.63; Cmax = 478 ± 111 μg/mL and 451 ± 74; half-life = 36 ± 9 min and 28 ± 9 for the micelle formulation and Restasis, respectively. The ocular distribution investigation revealed that the novel formulation delivered 1540 ± 400 ng CyA/g tissue to the cornea. CONCLUSIONS: The micelle formulation delivered active CyA into the cornea without evident negative influence on the ocular surface properties. This formulation could be applied for immune-related ocular surface diseases.