Development and application of a scintillation proximity assay (SPA) for identification of selective inhibitors of 11beta-hydroxysteroid dehydrogenase type 1.

Pre-receptor metabolism of glucocorticoids by the 11beta-hydroxysteroid dehydrogenase (11betaHSD) enzymes has been implicated in the etiology of the metabolic syndrome. Recent studies have shown that alterations in the activity of the type 1 isozyme can affect many aspects of the disease. This paper describes the optimization and application of a high-throughput scintillation proximity assay (SPA) developed to identify selective specific inhibitors of 11betaHSD1. Microsomes containing 11betaHSD1 were incubated in the presence of NADPH and [3H]cortisone, and the product, [3H]cortisol, was specifically detected in the mixture by a monoclonal antibody coupled to protein A-coated SPA beads with greater than 2 log higher affinity for cortisol than cortisone. Dimethyl sulfoxide and NADPH co-substrate additions were optimized for 11betaHSD1 reductase activity. Titrated test compound, when introduced into the optimized assay, reproducibly inhibited the enzyme and yielded consistent IC50 data in either 96- or 384-well format. An 11betaHSD2 counterscreen was performed by incubating 11betaHSD2 microsomes with [3H]cortisol and NAD+ and monitoring substrate disappearance.

Relationship of calf antibody status to disease and performance.

Three tests were used to measure the circulating immunoglobulin in 381 purchased calves as they entered a commercial calf-rearing unit. A correlation of 0.64 was found between the zinc sulphate turbidity (ZST) test and a quantitative latex agglutination test (LAT) measuring IgG1 (P less than 0.001). A qualitative version of the LAT related poorly to the quantitative version. The proportion of plasma samples identified by the quantitative LAT as having an IgG1 concentration of less than 5 g/litre which were incorrectly identified as positives (greater than or equal to 5 g/litre) by the qualitative LAT was 0.65. The proportion of plasma samples identified by the quantitative LAT as having a IgG1 concentration of greater than or equal to 5 g/litre which were incorrectly identified as negative (less than 5 g/litre) was 0.11. There was no statistically significant relationship between plasma IgG1 concentration and initial liveweight, subsequent overall daily liveweight gain or disease incidence (P greater than 0.05). Calves treated for infectious disease, particularly respiratory disease after weaning, had statistically significantly lower liveweight gains than healthy calves (P less than 0.001).

Migration of xenogenic astrocytes in myelinated tracts: a novel probe for immune responses in white matter.

Experimental brain transplantation allows the study of the development of the immune response against brain antigens within the brain itself. This laboratory has developed a transplantation model in which rabbit embryo brain fragments are placed in the brains of newborn mice. The migration of xenogenic astrocytes is traced by a monoclonal antibody which combines with donor but not host glial fibrillary acidic protein. In the first 4 weeks after transplantation, the donor astrocytes successfully migrate, often within myelinated tracts. Following this period, T cells make their appearance and xenogenic astrocytes disappear by 10 weeks. The propensity for clearly identified foreign astrocytes to migrate in myelinated tracts coupled with a well-defined time course of host-vs-graft interaction suggested that the model could be used to study the immune response in white matter. The studies reported here provide sequential examples of the relationship between migration by foreign astrocytes in myelinated tracts and the development of the host immune response. Extensive migration in white matter tracts was first observed in the absence of any T cell response. Subsequently T cells were found at the transplantation site. Finally Ia was found to be expressed on blood vessels and microglia were strongly reactive in white matter that contained T cells but no foreign astrocytes. These observations support the suggestion that the model can be used to more precisely define cellular immune events that occur within white matter.