RESUMO
The focus of animal welfare science has shifted over the last decades from efforts to avoid negative states to ways of allowing animals the experience of positive emotions. They may influence physiological processes in farmed animals, potentially providing health benefits; in addition, the physiological changes might be used as indicators of emotional states. We investigated calves' salivary secretory immunoglobulin A (sIgA) concentrations with regard to a possible circadian rhythm and two situations that elicit positive emotions. Ten saliva samples of 14 calves were taken on two consecutive days; within the course of a day we observed a significant decline in salivary sIgA concentrations at 14:00 h. Further, we probed the animals before and after milk feeding and, contrarily to our prediction, detected lower sIgA concentrations 5 min after feeding than 15 min before. A probable explanation might be an increase in salivary flow rate caused by milk ingestion. We also took samples before and after we stimulated play behavior in calves. There was no significant difference in sIgA concentrations between samples taken before and after play. Although there was a significant correlation between the change in sIgA concentrations and the amount of play behavior shown, the correlation depended on an unexpected decrease of sIgA in animals that played little, and thus, does not support our hypothesis. In general, the data showed a large variability that might arise from different factors that are difficult to standardize in animals. Thus, the use of salivary sIgA concentrations as a marker of positive emotions in calves is not supported conclusively by the present data.
RESUMO
BACKGROUND: 30 years ago, the potential of bispecific antibodies to engage cytotoxic T cells for the lysis of cancer cells was discovered. Today a variety of bispecific antibodies against diverse cell surface structures have been developed, the majority of them produced in mammalian cell culture systems. Beside the r28M, described here, no such bispecific antibody is known to be expressed by transgenic livestock, although various biologicals for medical needs are already harvested-mostly from the milk-of these transgenics. In this study we investigated the large-scale purification and biological activity of the bispecific antibody r28M, expressed in the blood of transgenic cattle. This tandem single-chain variable fragment antibody is designed to target human CD28 and the melanoma/glioblastoma-associated cell surface chondroitin sulfate proteoglycan 4 (CSPG4). RESULTS: With the described optimized purification protocol an average yield of 30 mg enriched r28M fraction out of 2 liters bovine plasma could be obtained. Separation of this enriched fraction by size exclusion chromatography into monomers, dimers and aggregates and further testing regarding the biological activity revealed the monomer fraction as being the most appropriate one to continue working with. The detailed characterization of the antibody's activity confirmed its high specificity to induce the killing of CSPG4 positive cells. In addition, first insights into tumor cell death pathways mediated by r28M-activated peripheral blood mononuclear cells were gained. In consideration of possible applications in vivo we also tested the effect of the addition of different excipients to r28M. CONCLUSION: Summing up, we managed to purify monomeric r28M from bovine plasma in a large-scale preparation and could prove that its biological activity is unaffected and still highly specific and thus, might be applicable for the treatment of melanoma.
