Monodisperse magnetic polymer particles. New biochemical and biomedical applications.

The method of activated swelling of polymer particles developed by the authors allows the preparation of monodisperse spherical beads of predictable size from 1 to 100 microns in diameter. The polymer particles may be prepared from a number of different monomeric materials and with various morphologies including macroporous structures. The porous beads form the basis for magnetizable monodisperse polymer particles which have magnetic iron oxides distributed as small grains all through the volume of the beads. The magnetic particles are being used extensively for selective cell separation and for immunomagnetic separation within microbiology and molecular biology. A review of recent work within these fields is given. New methods for positive cell separation are announced.

Monosized, magnetic polymer particles: their use in separation of cells and subcellular components, and in the study of lymphocyte function in vitro.

By employing the principles of "activated swelling", monosized, superparamagnetic polymer particles have been prepared ranging in size from 1-100 microns. Both during and after the swelling process, the particles can be modified to meet a series of specific demands making them potentially very interesting for many separation and assay purposes. Using monoclonal antibodies to direct the magnetic beads to their targets, immunomagnetic separation has turned out to be one of the most specific, reliable and, above all, the fastest technique available today to isolate particulate material for further studies. So far, most efforts have been concentrated on methodology for fractionation of cells in suspension, such as removal of tumour cells from bone marrow or isolation of lymphoid cells from peripheral blood. These studies have both established the parameters necessary for optimal performance and at the same time laid the groundwork for future developments making immunomagnetic separation an exciting new tool in many research areas. High speed and specificity are the most conspicuous features of immunomagnetic cell separation. These properties have been exploited in the successful development of a new technique for tissue typing of cells directly from peripheral blood specimens. Both higher sensitivity and specificity have been obtained. The same principles can be used for fast and safe quantification of cell populations and subpopulations in blood and cell suspensions. The functions of, and interactions between, peripheral blood cell populations or subpopulations in the immune response have also been studied with high precision. The significance of direct cell contact on the one hand, and soluble factors on the other, can now be established in detail. Immunomagnetic beads have also been used to study the interaction between various T lymphocyte membrane molecules in the early phases of the activation process. Finally, the usefulness of specially developed particles for the fractionation of subcellular components is described.

Ileal metabolism in vitro of testosterone to 4-androstene-3 alpha, 17 beta-diol.

Testosterone metabolism to 4-androstene-3 alpha, 17 beta-diol by the 800 g supernatant fraction of ileum from male and female rats was investigated. Ileal production of this testosterone metabolite was higher in mature female animals than in mature males. This difference could be eliminated by administration of large doses estradiol-17 beta to mature male rats. Immature animals showed low ileal production of 4-androstene-3 alpha, 17 beta-diol before being weaned from their mother.

Metabolism in vitro of testosterone (T) to 17 beta-hydroxy-5 alpha-androstane-3-one (DHT) and 5 alpha-androstane-3 alpha,17 beta-diol (3 alpha) by the 800 g supernatant fraction of ileum from rats.

The 800 g supernatant fraction of ileum from rats was incubated with testosterone and production of 17 beta-hydroxy-5 alpha-androstane-3-one and 5 alpha-androstane-3 alpha,17 beta-diol measured. No difference in this production could be determined between ileum from mature female and mature male animals. Ileum from immature rats staying with their mothers from day 12 to day 21 showed increased 5 alpha-reduction of testosterone on days 15, 17 and 21. Ileal metabolism to 17 beta-hydroxy-5 alpha-androstane-3-one and 5 alpha-androstane-3 alpha,17 beta-diol was increased in immature animals on day 25 of extrauterine life when consuming rat chow and water ad libitum from day 21. This increase could not be demonstrated on day 28. Immature and mature animals treated either with long-acting ACTH or dexamethasone showed increased ileal conversion of testosterone to the two 5 alpha-reduced metabolites determined. Ileum from mature male rats injected, subcutaneously, with arabinosylcytosine every 8th h for 2 days exhibited increased metabolism of testosterone to 17 beta-hydroxy-5 alpha-androstane-3-one and 5 alpha-androstane-3 alpha,17 beta-diol from the 1st to the 6th day after the last injection of arabinosylcytosine. Different aspects of intestinal metabolism of testosterone are discussed.

In vitro metabolism of testosterone to 17 beta-hydroxy-5 alpha-androstane-3-one and 5 alpha-androstane-3 alpha, 17 beta-diol by the rat intestine.

The metabolism of testosterone to 17 beta-hydroxy-5 alpha-androstane-3-one and 5 alpha-androstane-3 alpha, 17 beta-diol by the 800 g supernatant fraction by different parts of the gastrointestinal tract from male rats was investigated. This metabolism tended to be higher in immature than in mature animals. Administration of dexamethasone or long-acting ACTH to immature and mature rats increased testosterone metabolism to 17 beta-hydroxy-5 alpha-androstane-3-one and 5 alpha-androstane-3 alpha, 17 beta-diol by ileum tissue. No such effect could be observed following administration of progesterone, estradiol, prolactin, LH or FSH in mature animals. Development of the gastrointestinal tract from the immature to the mature stage was associated with augmented metabolism of testosterone to 17 beta-hydroxy-5 alpha-androstane-3-one and 5 alpha-androstane-3 alpha, 17 beta-diol in the ileum.

Androgen metabolism in rat skeletal muscle in vitro.

Androgen metabolism by the cytosol fraction of rat skeletal muscle was investigated. Testosterone metabolism was low, the main metabolite being 4-androstene-3 alpha,17 beta-diol. In addition, small amounts of 5 alpha-androstane-3 alpha, 17 beta-diol were formed, but no 17 beta-hydroxy-5 alpha-androstane-3-one could be detected. 4-Androstene-3 alpha,17 beta-diol was metabolized only to testosterone in this system of incubation. When 17 beta-hydroxy-5 alpha-androstane-3-one was incubated with muscle cytosol, considerable metabolism to 5 alpha-androstane-3 alpha,17 beta-diol and to 5 alpha-androstane-3 beta,17 beta-diol could be detected. Low 5 alpha-reduction of testosterone and rapid conversion of formed 17 beta-hydroxy-5 alpha-androstane-3-one to 5 alpha-androstane-3 alpha,17 beta-diol and 5 alpha-androstane-3 beta,17 beta-diol gave limited ability of the muscle preparation employed to accumulate 17 beta-hydroxy-5 alpha-androstane-3-one.

Androgen metabolism in relation to growth stimulation by a uterine cell line.

The metabolism of radioactive testosterone, 5alpha-dihydrotestosterone, 4-androstene-3beta,17beta-diol or 4-androstene-3alpha,17beta-diol by the human cell line NHIK 3025, derived from a carcinoma of the uterine cervix, was studied. The cells were grown in Eagle's minimal essential medium (MEM) with a steriod concentration of 10-(7) M for 4 days. Androgen metabolism by this cell line is essentially the same as for other androgen-responsive cells. The most interesting testosterone metabolite in this system is 4-androstene-3beta,17beta-diol, and the separation of this compound from 4-androstene-3alpha,17beta-diol and the two corresponding 5alpha-reduced diols is described. Since 4-androsterone-3beta,17beta-diol is a more potent growth factor for these cells than testosterone, the small conversion of testosterone to 4-androstene-3beta, 17beta-diol observed could be responsible for the growth stimulation by testosterone.