Quantitative comparison of click beetle and firefly luciferases for in vivo bioluminescence imaging.

For bioluminescence imaging (BLI) of small animals, the most commonly used luciferase is Fluc from the firefly, but recently, green (CBGr99) and red (CBRed) click beetle luciferases became available. Because signal attenuation by tissues is lower for red light, red luciferases appear to be advantageous for BLI, but this has not been thoroughly tested. We compare different luciferases for BLI. For this purpose, cell transfectants are generated expressing comparable amounts of CBGr99, CBRed, or Fluc. This is achieved by coexpression of the luciferase with eGFP using the bicistronic 2A system, which results in stoichiometric coexpression of the respective proteins. In vitro, the CBGr99 transfectant exhibits the strongest total photon yield. For in vivo BLI, the transfectants are injected into mice at different locations. At a subcutaneous position, CBGr99 is clearly superior to the other luciferases. When the tumor cells are located in the peritoneum or lung, where more absorption by tissue occurs, CBGr99 and CBRed transfected cells emit a comparable number of red photons and are superior to Fluc, but CBGr99 reaches the maximum of the light emission faster than CBRed. Thus, although CBGr99 emits mainly green light, the high yield of total and red photons makes it an excellent candidate for BLI.

Functional significance of non-neuronal acetylcholine in skin epithelia.

In recent years, the physiological role of non-neuronal acetylcholine (ACh) and its receptors (AChR) in epidermal physiology has been under intense investigation. However, little is known about the role of the non-neuronal cholinergic system in inflammatory skin diseases. We chose the clinically nicotine-dependent skin disease hidradenitis suppurativa (HS) as model to study the influence of long term nicotine ingestion on epidermal morphology and AChR expression. HS is a chronic inflammatory, disabling disease of unknown pathogenesis emerging from the pilosebaceous unit of the intertriginous areas. In order to correlate our findings to specific nicotine effects, we used the organotypical coculture system (OTC) and raised artificial epidermis in the presence of nicotine. After 12 days in culture control OTC showed a mature epithelium, while nicotine treated OTCs were significantly thicker. Using immunofluorescence analysis, nicotine treated OTCs produced significantly stronger immunoreactivity (IR) for the alpha3, M(3) and M(5) AChR antisera than control. In contrast, the alpha7 nAChR antiserum showed a slightly reduced IR in the granular layer and the alpha9 nAChR IR retracted to the lower suprabasal layers. In HS epidermis we found the strongest IR for all AChR around the follicular infundibulum while in the sinus epithelia it was only weak. In contrast to the nicotine treated OTC, the alpha7 nAChR IR in the hyperplastic HS epidermis was clearly extended to all living layers. Altogether we provide first hints for a causative role of the non-neuronal cholinergic system in the pathogenesis of HS by promoting infundibular epithelial hyperplasia and thus follicular plugging.