The Ly-6 locus encodes several cell surface proteins of 10–12 kDa. Some members of this multigene family may function in cell signaling and/or cell adhesion processes. T lymphocytes overexpressing Ly-6A.2 (one member of the Ly-6 gene family) protein homotypically aggregate when cultured in vitro. Further analysis of this homotypic aggregation suggests that Ly-6A.2 participates in cell-cell adhesion. These observations indicated the presence of a Ly-6 ligand(s) on the surface of lymphoid cells. In this study we report generation of a hamster mAb, 9AB2, that blocks Ly-6A.2-dependent cell-cell adhesion. The 9AB2 Ab recognizes a 66-kDa glycoprotein with unique tissue expression. The 9AB2 mAb does not bind Ly-6A.2, but coimmunoprecipitates Ly-6A.2 molecule. Moreover, 9AB2 Ag-expressing thymocytes specifically bind to Chinese hamster ovary cells overexpressing Ly-6A.2 protein, and this binding is specifically blocked by 9AB2 and anti-Ly-6A.2 Abs. These results suggest that the 66-kDa protein recognized by 9AB2 mAb is the putative ligand for Ly-6A.2.

The preparation and topical antiinflammatory potencies of a series of halomethyl 17 alpha-(acyloxy)- 11 beta-hydroxy-3-oxoandrosta-1,4-diene-17 beta-carbothioates, carrying combinations of 6 alpha-fluoro, 9 alpha-fluoro, 16-methyl, and 16-methylene substituents, are described. Key synthetic stages were the preparation of carbothioic acids and their reaction with dihalomethanes. The carbothioic acids were formed from 17 beta-carboxylic acids by initial reaction with dimethylthiocarbamoyl chloride followed by aminolysis of the resulting rearranged mixed anhydride with diethylamine, or by carboxyl activation with 1,1'-carbonyldiimidazole (CDI) or 2-fluoro-N-methylpyridinium tosylate (FMPT) and reaction with hydrogen sulfide, the choice of reagent being governed by the 17 alpha-substituent. Carboxyl activation with FMPT and reaction with sodium hydrogen selenide led to the halomethyl 16-methyleneandrostane-17 beta-carboselenoate analogues. Anti-inflammatory potencies were measured in humans using the vasoconstriction assay and in rats and mice by a modification the Tonelli croton oil ear assay. Best activities were shown by fluoromethyl and chloromethyl carbothioates with a 17 alpha-propionyloxy group. S-Fluoromethyl 6 alpha, 9 alpha-difluoro-11 beta-hydroxy-16 alpha-methyl-3-oxo-17 alpha- (propionyloxy)androsta-1,4-diene-17 beta-carbothioate (fluticasone propionate, FP) was selected for clinical study as it showed high topical antiinflammatory activity but caused little hypothalamic-pituitary-adrenal suppression after topical or oral administration to rodents.

The ability of normal mouse mammary epithelial cells (MECs) to express insulin-like growth factor-binding proteins (IGFBPs) was examined. MECs were isolated from day 11 pregnant mice and cultured on floating collagen gels in serum-free basal medium. After 24 h, the medium was replaced with fresh medium with/or without mouse PRL (mPRL), mouse placental lactogen-I (mPL-I), mPL-II, mouse GH (mGH), IGF-I, and IGF-II, either alone or in combinations. The MECs were cultured for an additional 5 days before collection of conditioned medium (CM). The relative amount of IGFBPs present in the CM was determined by Western ligand blotting, and alpha-lactalbumin content was determined with a specific RIA. The CM of the MECs contained two IGFBPs, with approximate mol wt of 29K and 40-45K. The 40-45K IGFBP appears to be the mouse equivalent of IGFBP-3, but the identity of the 29K IGFBP is not presently known. The 29K IGFBP was not N-glycosylated and did not cross-react with antiserum to rodent IGFBP-2 or human IGFBP-1. Basal IGFBP expression was very low, but the addition of mPL-I, or mPL-II stimulated a marked increase in the amount of 29K IGFBP that was released into the CM and a lesser increase in the release of IGFBP-3. This increase in the release of 29K IGFBP was dose dependent, with increases found at concentrations as low as 1 ng/ml lactogen. mGH also stimulated the release of 29K IGFBP, but was less potent than any of the three lactogens. Treatment of MECs with either IGF-I or IGF-II increased the amount of both the 29K IGFBP and IGFBP-3 in the CM, with relative potencies similar to those of the lactogenic hormones. However, when either IGF-I or IGF-II was added together with one of the lactogenic hormones, the release of 29K IGFBP was increased in an additive manner. While the IGFs acted additively with the lactogenic hormones on the expression of 29K IGFBP, they did not stimulate alpha-lactalbumin production by the MECs or act to enhance the effects of the lactogenic hormones in stimulating alpha-lactalbumin production. This study demonstrates that IGFBPs are expressed in normal mouse MECs, and the release of these IGFBPs into the CM is hormonally regulated by both lactogenic hormones and IGFs.

The TCRs expressed on T lymphocytes recognize foreign peptides bound to MHC molecules. This reactivity is the basis of specific immune response to the foreign Ag. How such specificities are generated in the thymus is still being debated. Signals generated through TCR upon interaction with self MHC-peptide complexes are critical for maturation of the CD4+ helper and CD8+ cytotoxic subsets. We have observed maturation of CD4+ but not CD8+ T cells in Ly-6A.2 transgenic MHC null mice. Since there can be no interactions with MHC molecules in these mice, these CD4+ cells must express the T cell repertoire that exists before positive and negative selection. Interestingly, despite an absence of selection by MHC molecules, the CD4+ cells that mature recognize MHC molecules at a frequency as high as in CD4+ cells in normal mice. These results demonstrate that: 1) the germline sequences encoding TCRs are biased toward reactivity to MHC molecules; and 2) CD4+ cells as opposed to CD8+ cells have distinct lineage commitment signals. These results also suggest that signals originating from Ly-6 can promote or substitute for signals generated from TCR that are required for positive selection. Moreover, this animal model offers a system to study T cell development in the thymus that can provide insights into mechanisms of lineage commitment in developing T cells.

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