Edward J. Roy, PhD - US grants

Reproductive hormones influence reproductive behaviors 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 neurochemical processes by which hormones affect behavior. Our model system for this purpose is the influence of estrogens on mating behavior 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.

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.

Glucocorticoids, a family of steroid hormones secreted primarily by the adrenal glands, bind selectively to protein receptors in the hippocampus, a brain region involved in the formation of new memories. Both the pyramidal cells of the CA1 region and granule cells of dentate gyrus within the hippocampal structure show extensive hormone binding, much higher than the rest of the brain. These steroids are normally secreted at basal levels unless elevated during times of stress. Chronic elevations of physiological glucocorticoids, for example during prolonged stress, have been shown to have deleterious effects on pyramidal cells of the hippocampus and this is alleviated by removal of the adrenals. Long-term adrenalectomy is not without cost to the hippocampus, however, since this treatment causes loss of granule cells in the dentate gyrus. Thus, in selective regions of the hippocampus, basal levels of glucocorticoids actually protect the cells from dying. Dr. Roy will characterize the anatomical changes that occur to the dentate gyrus following adrenalectomy. Furthermore, since this is a critical brain region for learning and memory, he will correlate changes in the anatomy with performance in tests of spatial memory. The results will not only yield vitally significant information about the basic function of glucocorticoids in neuronal survival and cell death, but will also define its behavioral consequences when this protective effect of hormones is removed. Information about the basic processes underlying the death of subpopulations of brain cells as well as the mechanisms that prevent cell death in a healthy brain may impact greatly on our appreciation and treatment of such brain disorders as caused by stroke, Alzheimer's disease, and the normal aging process.

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.

[unreadable] DESCRIPTION (provided by applicant): [unreadable] Like antibodies, T cell antigen receptors (TCRs) recognize a huge diversity of molecular targets. The range of cancer related targets that T cells can recognize is even greater than what antibodies recognize, because intracellular proteins, such as transcription factors and other proteins that are modified in cancer, are processed and presented on the cell surface as peptide/MHC complexes that T cells recognize, whereas antibodies recognize only proteins that function on the cell surface. In addition, T cells have properties of active mobility that allow them to penetrate the blood brain barrier and migrate through solid tumors that antibodies will not readily penetrate. Phase I will test two ideas: (1) because iron strongly alters MRI signals, a clonal population of T cells loaded with iron nanoparticles can be used as an imaging agent in cancer, particularly brain tumors. Only tumor cells expressing a particular tumor associated peptide will be identified by the T cells. (2) Certain iron nanoparticles generate intense heat when exposed to alternating magnetic fields; this property can be used therapeutically. "Suicide bomber" T cells loaded with iron nanoparticles will specifically bind to tumor cells. Once their presence in the tumor is confirmed by MRI, alternating magnetic fields can be applied to produce damage in the surrounding cells, generating heat shock proteins and tumor antigens that will instigate a stronger immune response. Phase II tests two ideas: (1) the immune response generated by heating intratumor iron will be greatly enhanced if combined with danger signals, such as CpG oligonucleotides, that increase antigen presentation in lymph nodes. (2) Both of these uses of iron loaded T cells, imaging and therapy, will be enhanced by transfecting T cells with genetically engineered T cell receptors with higher affinity for tumor associated epitopes. High affinity T cell receptors for a model tumor antigen have been generated by yeast display technology, and experiments will determine if these high affinity T cell receptors enhance the imaging and therapeutic potential of iron loaded T cells. [unreadable] [unreadable]