NK cells detect changes in adaptive immunity within mouse decidua from gestation day eight.

Viable human CD56+ CD16- peripheral blood Natural Killer (NK) cells show specific in vitro binding under shear forces to ligands expressed by endothelial cells in cryostat sections of gestation day (gd)7 mouse decidua basalis. In serial assays, numbers of cells adhering to gd7 tissue are constant for men but have cyclical variation for fertile women, suggesting a brief gain in functional decidual homing potential of this NK cell subset during the menstrual cycle. Regardless of gender, numbers of adhering cells from an individual donor, increase dramatically when the substrate is decidua basalis from a later gestational timepoint. Here, we report that human blood CD56+ CD16- NK cells which adhere as single cells over gd7 decidua basalis, adhere as large clusters over gd8 and gd9 tissues, suggestive of antigen recognition and lymphocyte activation. We asked which cells within mouse decidua basalis trigger this response in CD56+ CD16- cells. Using decidua from mice transgenic for myeloid dendritic cell (mDC) expression of enhanced yellow fluorescent protein (eYFP), we found cluster formation was independent of mDC contact. Use of decidua from alymphoid mice showed clustering behavior required substrate lymphocytes. By use of decidua containing NK cells but lacking T and B cells, decidual T and/or B lymphocytes were identified as the cells altered after gd7 in a manner that activates CD56+ CD16- cell clustering. This timepoint is just prior to mouse spiral arterial modification and its detection by these indicator cells implicates adaptive, decidual immune responses in the regulation of NK cell function.

The international validation of a fixed-dose procedure as an alternative to the classical LD50 test.

In an international study involving 33 laboratories in 11 countries, the acute oral toxicity to the rat of 20 substances and preparations was evaluated using a fixed-dose procedure and the results compared with those obtained for the test materials using the classical LD50 test. The study has shown that the fixed-dose approach to acute oral toxicity testing: (1) produces consistent results that are not substantially affected by inter-laboratory variations; (2) provides adequate information for risk assessment purposes on signs of toxicity, including their nature, time to onset, duration and outcome; (3) uses fewer animals than the current internationally agreed OECD procedure (Guideline 401-revised); (4) subjects animals to less pain and distress than the classical LD50 test and causes less compound-related mortality; and (5) enables substances and preparations to be ranked according to the EEC classification system on the basis of their acute oral toxicity, such ranking being compatible with that allocated by the results of classical LD50 studies.

Acceptance of in vitro testing by regulatory authorities.

National regulatory authorities have responsibility for taking decisions that possibly affect the health of whole populations and it is therefore to be expected that they will be reluctant to substitute alternative in vitro toxicity test methods for conventional animal studies unless the new procedures have been demonstrated to be reliable and have been adequately validated. Validation of in vitro methods presents particularly difficult problems because whilst they tend to produce consistent and objective results, the test systems used are incapable of mirroring the complexity of the biochemical processes seen in animals. As a result, a single animal study would need a large battery of in vitro studies to replace it to cover the various endpoints that are involved in the in vivo study; each of these in vitro tests would need to be validated with regard to the specific endpoint that it is investigating. Although great advances have been made in recent years in the development of alternative methods, few have been validated to an extent that makes them acceptable to regulatory authorities as replacements for in vivo studies. Rather, they are largely seen and used as screening techniques whereby decisions can be taken on the value of further development of newly discovered compounds, and as aids in the interpretation of animal studies and in their extrapolation to man, that is, they are of value in 'mechanism of action' studies. Nevertheless, certain in vitro procedures are already accepted by regulatory authorities and their use, for example, in 'screening out' compounds that have severe irritant properties, and in identifying compounds with potential mutagenic and carcinogenic activity, has had a profound effect on both the number of animal studies carried out and on the welfare of those animals still used.

Evaluation of the BTS approach to the testing of substances and preparations for their acute toxicity.

This study has shown that the new approach to acute oral toxicity testing proposed by the British Toxicology Society: provides the information on the toxic effects of materials necessary for human risk assessment, can be used to rank materials for regulatory purposes on the basis of their acute toxicity, provides advantages for animal welfare by requiring the use of fewer animals than conventional acute oral toxicity tests and by subjecting those animals used to less chance of experiencing pain or distress.