Kathrin M. Scherer

Failure of omalizumab (Xolair®) in the treatment of a case of solar urticaria caused by ultraviolet A and visible light

Solar urticaria is a rare photodermatosis probably caused by a chromophore, that – if activated by light of a specific spectrum – binds to mast cell‐bound IgE and elicits degranulation. In our patient an action spectrum in ultraviolet A and visible light range was found, in the autologous serum test the presence of a serum chromophore for the same action spectra could be demonstrated, which may underline this pathogenetic hypothesis. Symptoms did not improve using antihistamines and sun protection. Photo hardening was denied from the patient, immunosuppression and plasmapheresis were discussed but not considered. So a treatment with Omalizumab was started that recently was successfully used in 4 case reports. After 3 doses of Xolair® there was no changing in the phototesting results and after 4 doses no subjective improvement.

Contact dermatitis to para-phenylenediamine in hair dye following sensitization to black henna tattoos - an ongoing problem.

Summary  Background: The increased frequency of case reports of allergic contact dermatitis from non‐permanent black henna tattoos in recent years shows the popularity of this form of body painting.

Hypersensitivity to proton pump inhibitors: diagnostic accuracy of skin tests compared to oral provocation test.

For comparison, lung tissue expressed relatively low levels of gap junction and desmosomes and some are fully not detectable (Fig 2,C). Normal human bronchial epithelial cells cultured inALI expressed claudin-17 (P 5 .03) (Fig 2, A). In contrast, primary keratinocyte in ALI cultures expressed uniquely desmocollin-1 (P 5 .02) (Fig 2, D). Keratinocyte ALIs did not express any TJ proteins in addition to the common junctional proteins observed in skin biopsies and monolayer cultures (Fig 2, B). Detailed statistical analysis is shown in Table E1 (in the Online Repository available at www.jacionline.org). Our data demonstrate that monolayer cell cultures express a similar profile of junctionmolecules compared with ALI cultures, however, with relatively low levels. After apicobasal differentiation in ALIs, more junctional molecules are expressed (Fig 2). Interestingly, whole tissues expressed a much broader profile of junctional molecules. The main reason behind having additional junctional mRNAs appearing in the whole tissue of lung and skin can be that other cell types, such as endothelial cells, fibroblasts in both, and smooth muscle cells in the lung, are also expressing junctional molecules. ALIs represent here a more cell-type–specific junctional molecule expression pattern. After performing the detailed expression analyses of interepithelial barrier molecules, we realized that it is essential to study the TJs in a broader way. Although some are dominantly expressed, the determination of one or two junctional molecules, especially TJs, may not represent the whole picture. There are distinct and overlapping patterns of TJs and other cell-cell adhesion molecules expressed in the skin and lung epithelia, which implicate a highly regulated and complex pattern. Each cell type shows its own, unique expression profile reflecting its specialization related to its function within the organism. In addition, we have to take into account that TJs are not uniquely expressed in epithelial cells but are also found and regulated in other cells, especially in endothelium. Furthermore, other mesenchymal tissues also express them as recently observed in smooth muscle cells in asthma. The number of different junctional proteins, which are expressed, interact in multiple ways in every cell type to form appropriate cell-cell contacts and tissue integrity. Our data demonstrated here suggest that the picture of the junctional apparatus in each cell type and tissue is distinct, and the regulation of epithelial integrity might be more complex than being considered so far. Jeannette I. Kast, BSc Kerstin Wanke, MSc Michael B. Soyka, MD Paulina Wawrzyniak, MSc Deniz Akdis, BSc K€ ulli Kingo, MD, PhD Ana Rebane, PhD Cezmi A. Akdis, MD

Fluorescence lifetime imaging of E-combretastatin uptake and distribution in live mammalian cells

To investigate within live mammalian cells the uptake and disposition of combretastatins, fluorescence lifetime imaging was used with two-photon excitation (2PE). Combretastatin A4 (CA4) and analogues are potential anticancer drugs due to their ability to inhibit angiogenesis. E(trans)-combretastatins are considerably less active than the Z(cis)-combretastatins proposed for clinical use. However the E-combretastatins exhibit stronger intrinsic fluorescence with quantum yields and lifetimes that depend markedly on solvent polarity and viscosity. It is proposed that 2PE in the red and near-infrared tissue window may allow in situ isomerization of E-combretastatins to the more active Z-isomer, offering spatial and temporal control of drug activation and constitute a novel form of photodynamic therapy. In the present work we have characterised 2PE of E-CA4 and have used fluorescence lifetime imaging with 2PE to study uptake and intracellular disposition of E-CA4 and an analogue. The results show that these molecules accumulate rapidly in cells and are located mainly in lipidic environments such as lipid droplets. Within the droplets the local concentrations may be up to two orders of magnitude higher than that of the drug in the surrounding medium.

A series of flexible design adaptations to the Nikon E-C1 and E-C2 confocal microscope systems for UV, multiphoton and FLIM imaging

Multiphoton microscopy is widely employed in the life sciences using extrinsic fluorescence of low‐ and high‐molecular weight labels with excitation and emission spectra in the visible and near infrared regions. For imaging of intrinsic and extrinsic fluorophores with excitation spectra in the ultraviolet region, multiphoton excitation with one‐ or two‐colour lasers avoids the need for ultraviolet‐transmitting excitation optics and has advantages in terms of optical penetration in the sample and reduced phototoxicity. Excitation and detection of ultraviolet emission around 300 nm and below in a typical inverted confocal microscope is more difficult and requires the use of expensive quartz optics including the objective. In this technical note we describe the adaptation of a commercial confocal microscope (Nikon, Japan E‐C1 or E‐C2) for versatile use with Ti‐sapphire and OPO laser sources and the addition of a second detection channel that enables detection of ultraviolet fluorescence and increases detection sensitivity in a typical fluorescence lifetime imaging microscopy experiment. Results from some experiments with this setup illustrate the resulting capabilities.

Nanometric molecular separation measurements by single molecule photobleaching.

Although considerable progress has been made in imaging distances in cells below the diffraction limit using FRET and super-resolution microscopy, methods for determining the separation of macromolecules in the 10-50 nm range have been elusive. We have developed fluorophore localisation imaging with photobleaching (FLImP), based on the quantised bleaching of individual protein-bound dye molecules, to quantitate the molecular separations in oligomers and nanoscale clusters. We demonstrate the benefits of using our method in studying the nanometric organisation of the epidermal growth factor receptor in cells.

Three-dimensional imaging and uptake of the anticancer drug combretastatin in cell spheroids and photoisomerization in gels with multiphoton excitation

Abstract. The uptake of E-combretastatins, potential prodrugs of the anticancer Z-isomers, into multicellular spheroids has been imaged by intrinsic fluorescence in three dimensions using two-photon excited fluorescence lifetime imaging with 625-nm ultrafast femtosecond laser pulses. Uptake is initially observed at the spheroid periphery but extends to the spheroid core within 30 min. Using agarose gels as a three-dimensional model, the conversion of Z(trans)→E(cis) via two-photon photoisomerization is demonstrated and the location of this photochemical process may be precisely selected within the micron scale in all three dimensions at depths up to almost 2 mm. We discuss these results for enhanced tissue penetration at longer near-infrared wavelengths for cancer therapy and up to three-photon excitation and imaging using 930-nm laser pulses with suitable combretastatin analogs.

Anticancer phototherapy using activation of E-combretastatins by two-photon–induced isomerization

Abstract. The photoisomerization of relatively nontoxic E-combretastatins to clinically active Z-isomers is shown to occur in solution through both one- and two-photon excitations at 340 and 625 nm, respectively. The photoisomerization is also demonstrated to induce mammalian cell death by a two-photon absorption process at 625 nm. Unlike conventional photodynamic therapy (PDT), the mechanism of photoisomerization is oxygen-independent and active in hypoxic environments such as in tumors. The use of red or near-infrared (NIR) light for two-photon excitation allows greater tissue penetration than conventional UV one-photon excitation. The results provide a baseline for the development of a novel phototherapy that overcomes nondiscriminative systemic toxicity of Z-combretastatins and the limitations of PDT drugs that require the presence of oxygen to promote their activity, with the added benefits of two-photon red or NIR excitation for deeper tissue penetration.

Ultrafast vibrational spectroscopic Studies on the photoionization of the α-Tocopherol analogue Trolox C

The initial events after photoexcitation and photoionization of α-tocopherol (vitamin E) and the analogue Trolox C have been studied by femtosecond stimulated Raman spectroscopy, transient absorption spectroscopy and time-resolved infrared spectroscopy. Using these techniques it was possible to follow the formation and decay of the excited state, neutral and radical cation radicals and the hydrated electron that are produced under the various conditions examined. α-Tocopherol and Trolox C in methanol solution appear to undergo efficient homolytic dissociation of the phenolic -OH bond to directly produce the tocopheroxyl radical. In contrast, Trolox C photochemistry in neutral aqueous solutions involves intermediate formation of a radical cation and the hydrated electron which undergo geminate recombination within 100 ps in competition with deprotonation of the radical cation. The results are discussed in relation to recently proposed mechanisms for the reaction of α-tocopherol with peroxyl radicals, which represents the best understood biological activity of this vitamin.

Spectroscopy and fluorescence lifetime imaging in live cells of a cyano-substituted combretastatin

Fluorescence lifetime imaging has been used to observe the real-time uptake in live mammalian cells of a combretastatin-type drug analogue that is a substituted stilbene with a cyano group at the bridging olefinic bond. Fluorescence spectra in a range of solvents show that this molecule has a substantial increase in dipole moment on promotion to the first excited singlet state that is indicative of intramolecular charge transfer (CT) from the methoxy substituents on the aromatic rings to the electron-withdrawing cyano group. This CT process is also demonstrated in picosecond time-resolved infrared measurements. Consistent with these observations, the molecule has been found to have a large two-photon absorption crosssection,and suggests potential routes to the design of a molecule that could be effectively activated by photoisomerisation in a two-photon process.