No abstract

Radiation causes soft tissue complications that include fibrosis and deficient wound healing. ␤-Catenin , a key component in the canonical Wnt-signaling pathway , is activated in fibrotic processes and wound repair and , as such , could play a role in mediating cellular responses to irradiation. ␤-Catenin can form a transcriptionally active complex with members of the Tcf family. A reporter mouse model , in addition to human cell cultures , was used to demonstrate that ionizing radiation activates ␤-catenin-mediated , Tcf-dependent transcription both in vitro and in vivo. Furthermore , radiation activates ␤-catenin via a Wnt-mediated mechanism , as in the presence of dickkopf-1 , an inhibitor of Wnt receptor activation , ␤-catenin levels did not increase after irradiation. Fibroblast cell cultures were derived from mice expressing either null or stabilized ␤-catenin alleles. Cells expressing stabilized ␤-catenin alleles had a higher proliferation rate and formed more colony-forming units than wild-type or null cells after irradiation. Wound healing was studied in these same mice after irradiation. There was a positive correlation between the tensile strength of the wound , the expression levels of type 1 collagen in the skin , and ␤-catenin levels. Mice treated with lithium showed increased ␤-catenin levels and increased wound strength. ␤-Catenin mediates the effects of ionizing radiation in fibroblasts , and its modulation has the potential to decrease the severity of radiation-induced soft tissue complications.

The physical stability of a monoclonal antibody (mAb) solution for injection in a pre-filled syringe may in part depend on its behaviour at the silicone oil/water interface. Here, the adsorption of a mAb (termed COE-3) and its fragment antigen-binding (Fab) and crystallisable (Fc) at the oil/water interface were measured using neutron reflection. A 1.4±0.1 µm hexadecane oil film was formed on a sapphire block by a Spin-Freeze-Thaw process in order to retain its integrity upon contact with the protein solutions. Measurements revealed that adsorbed COE-3, its Fab and Fc retained their globular structure, forming layers that did not penetrate substantially into the oil phase. COE-3 and Fc were found to adsorb flat-on to the interface, with a denser 45 and 42 Å inner layer, respectively, in contact with the oil and a more diffuse 17-21 Å outer layer caused by fragments adsorbing in a tilted manner. In contrast, Fab fragments formed a uniform 60 Å monolayer. Monolayers were formed under all conditions studied (10-200 ppm, using three isotopic contrasts), though changes in packing density across the COE-3 and Fc layers were observed. COE-3 had a higher affinity to the interface than either of its constituent fragments, while Fab had a lower interfacial affinity consistent with its higher net surface charge. This study extends the application of high resolution neutron reflection measurements to the study of protein adsorption at the oil/water interface using an experimental set-up mimicking the protein drug product in a siliconized prefilled syringe.