Follow-up study would be required to confirm cell specific effects. Such a follow-up study would likely need a larger sample size or narrower cell selection criteria. As we continue to run these experiments to test novel compounds with naproxen and oxycodone as positive controls and saline as a negative control, we will collate a larger dataset with which to investigate the effects on particular cell responses using larger sample sizes. In future studies this may allow us to set cell selection criteria and appropriate sample sizes to test if novel compounds target specific cell types or mechanisms. We are also performing an informal interim analysis for futility halfway through the experiment. When using such experiments as assays to discover novel therapies, this SCH727965 approach could result in a large reduction in animal use by stopping large studies that are very unlikely to produce positive evidence to support a hypothesis. This approach is in line with the guidelines of the National Centre for the Replacement, Refinement and Reduction of Animals in Research. Being guided by the ACT and the answers to its questions informed the design and interpretation of the follow-up experiment with naproxen. Infectious mastitis is defined as the inflammatory response initiated when microorganisms enter the mammary gland challenging the host defense. This common disease has either clinical or asymptomatic characteristics and is generally perceived as a significant burden for the well-being of mammals and especially dairy animals. Two main bacterial species that cause bovine mastitis are Escherichia coli and Staphylococcus aureus. Although both these pathogens grow in the mammary gland evoking a host immune response, they activate specific inflammatory signaling pathways which result in discriminatory stress profiles. This distinctive pathobiology can be explained by the microbeassociated molecular patterns dictating the expression and subsequent release of specific pro-inflammatory cytokines. In the initial phase, these mediators orchestrate the diapedesis of predominantly neutrophils into the mammary alveoli activating phagocytic innate immune cells to eliminate pathogens or at least prevent their spreading. Most of the observations seen in cows have been further elaborated at the molecular level through in vivo studies in mouse mastitis models. Our group previously reported that in vitro exposure of bovine neutrophils to live E. coli rapidly activates a complex series of molecular pathways involving cell death, the cleaving of the protease procaspase-1 and the transcription factor nuclear factorkappaB. This activity occurs concomitant with the secretion of the pro-inflammatory cytokine IL-1beta. Two studies from our group using intramammary infections in mice confirmed the relevance of these key parameters in vivo. However, elucidation of the link between these innate mammary host defense factors and their relevance for other mastitis pathogens than coliforms is just starting to emerge. It was already demonstrated for the bovine species that both E. coli and S. aureus bind mammary epithelial Toll-like receptor 4 and TLR2, but that both these pathogens differently modulate NFkappaB.