Edward J. Roy, PhD - US grants

Reproductive hormones influence reproductive behaviours and nonreproductive behaviors by acting directly in the central nervous system. The proposed experiments have as a long-term objective an increase in our understanding of the neurochemcial processes by which hormones affect behavior. Our model system for this purpose is the influence of estrogens on mating behaviour in the rat, because sexual receptivity in the female rat is completely contingent upon recent exposure of the brain to estrogen. The proposed studies will examine the importance of temporal parameters of stimulation by estrogen to test the hypothesis that there are two phases to estrogen action during which different neurochemical processes are stimulated. Other studies will characterize the regulation of the synthesis and degradation of receptor proteins for estrogen, since these proteins initiate the response to the hormone within neurons. Finally, studies on these problems will be conducted on tissue slices in vitro in order to determine what the specific neurochemical products of estrogen action are. Studies of hormone action in the brain are related not only to fundamental emotional and behavioral systems, but also to the control of the entire reproductive system, all areas relevant to health and disease.

antidepressants; aging; corticosteroids; chemical binding;

The goal of the research is an understanding of the basic neurochemical processes by which hormones affect behavior. Sexual behavior in the female rat is a convenient model, because the behavior is completely dependent on estrogen and progesterone, and previous work has established that an important site of these actions is the ventromedial hypothalamus. The most likely mechanism by which estrogen progesterone affect the behavior is a change in protein synthesis elicited by steroid receptors in cell nuclei. However, little is known about proteins that might be modified by either hormone, and alternative hypotheses have been proposed for progesterone's effects. the proposed experiments will directly test the hypothesis that progesterone modifies protein synthesis in the ventromedial hypothalamus of the female rat. labeled amino acid precursors will be infused into the ventromedial hypothalamus of ovariectomized rats. The patterns of proteins synthesized after control injections, estradiol alone, or estradiol plus progesterone will be analyzed using tow dimensional gel electrophoresis. The hypothesis predicts that there will be three different patterns of protein synthesis in rats which receive the different treatments. it is further hypothesized that some hormonally modified proteins will be transported to the midbrain central gray, and area known to be an important link in inducing sexual receptivity. The time course of progesterone's effect on protein synthesis will be investigated, and the ability of a progestin receptor antagonist to block the protein synthesis changes will assessed. Finding that progesterone modifies the synthesis of specific proteins in the brain would open a new avenue for studies of mechanisms by which hormones affect behavior.

DESCRIPTION: (Applicant's Abstract) In the past ten years, various strategies that use bispecific antibodies to redirect the activity to T cells against tumor cells have been developed. Despite the advance of several of these agents into clinical trials, there remain significant problems with the bispecific antibody approach. Chief among these problems has been the inability to sustain an active T cell infiltrate at the site of solid tumors. The overall goal of the proposed experiments is to sustain T cell activity at the site of the tumor and redirect the efficient lysis of tumor cells. Bispecific agents that target T cells to the high affinity folate receptor (FR), found on most ovarian carcinomas and some brain tumors, can be produced easily by attaching folate to any anti-T cell antibody of interest. This method will allow evaluation of the tumor-dependent T cell activating potential of various conjugates, including folate conjugates of antibodies to the T cell receptor/CD3 complex, CD28, and LFA-1. Multivalent ligation of these molecules on the surface of T cells stimulates full activation of T cells under normal physiological conditions. In contrast, ligation of the T cell molecule CTLA4 with its ligand B7 leads to T cell inactivation. Inhibition of the CTLA4:B7 interaction can sustain the activity of T cells. Based on these rationales the various folate/anti-T cell antibody conjugates will be tested alone and in combination with monovalent forms (scFv and Fab) of an anti-CTLA4 antibody. In Specific Aim 1, in vitro assays will measure T cell proliferation and cytotoxicity after incubation of T cells with folate/anti-TCR conjugates, anti-CTLA4 antibody fragments and FR tumor cells. In Specific Aim 2, mice treated in vivo with various antibody agents will be examined ex vivo for cytotoxicity by splenic T cells or peritoneal exudate cells and by immunohistochemical analysis of T cell infiltrates within tumors. The goal of this aim is to identify agents that optimally maintain activated T cells within the tumor. In Specific Aim 3, antibodies and/or folate-conjugates will be tested for their ability to eliminate FR+ tumors in three murine model systems: (a) immunologically defined 2C TCR/RAG mice transplanted with a human FR+ tumor; (b) immunocompetent mice transplanted with a syngeneic FR+ tumor; and (c) immunocompetent transgenic mice that develop endogenous FR+ brain tumors. Endpoints will include survival, tumor progression, and testing for tumor-specific memory by re-transplantation of the appropriate tumor.