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PILOT PROJECTSPilot 1, Year 1
"Genetically Modified Dendritic Cells as Vaccines for the Aged"
Abstract There is currently a paucity of effective treatments for metastatic prostate cancer, an important disease of the aged. One potential therapy that holds much promise is cytotoxic T lymphocyte (CTL)-based immunotherapy. Prostate cancer is an ideal target for immunotherapy, as all prostate carcinoma cells express prostate-specific antigen (PSA).There has recently been much interest in dendritic cells (DCs), the most potent antigen-presenting cell known, as a method of immunization for tumor immunotherapy due to their ability to elicit specific, powerful CTL responses. In order to manipulate DCs to express and process PSA for display on class I MHC molecules and presentation to CTL, we have generated herpes simplex virus-1 (HSV-1)-based amplicons. HSV-PSA amplicons are plasmid-based vectors that are packaged in HSV-1 capsids using a helper-free system, resulting in a viral vector that transduces PSA expression in the complete absence of viral gene products. Preliminary experiments indicate that DCs infected with HSV-PSA (or another model tumor antigen, ovalbumin) can present the expressed antigen to CTL and generate responses in vivo. As it is well known that the elderly have a decreased ability to generate immune responses, we seek to enhance DC function by expressing various cytokine genes in DCs as well as tumor antigens. The ability to transduce cells with multiple genes simultaneously is one of several unique benefits of the HSV-1 amplicon system. We will test the ability of such DCs to elicit anti-PSA CTL responses in normal as well as PSA transgenic mice, allowing us to account for immune tolerance mechanisms present in human males to PSA. We will also compare young mice to aged mice to determine whether additional cytokine expression can overcome the weakened immune response of the aged. Thus, the goal of this pilot proposal is to develop novel immunization methods using DCs and HSV-1 amplicons for the activation and enhancement of immune responses as a possible therapy for prostate cancer in aged patients. Such studies will allow us to address the hypothesis that by altering the cyokine profile of the APC that one can alter the magnitude and the quality of the immune response. Pilot 1, Year 2 "Effect of Gender on Neurogenesis in the Aging Brain"
Abstract The hippocampal dentate gyrus (DG) harbors neural precursor cells that continue to generate new neurons and glia into adulthood. Recent studies indicate that levels of adult DG neurogenesis are associated with memory function,but decrease with increasing age in female rodents, suggesting that age-related functional decline in the DG may be mediated by a decreased production of new neurons. However, because male rodents have not been included in these studies, it is not known whether males undergo age-related changes in adult neurogenesis. Since estrogen enhances DG neurogenesis in young adult female rodents, the decreased neurogenesis reported in postmenapausal females could be due to diminished estrogen levels (which would not be relevant to aging males) or instead, might reflect a general aging process that would affect males as well. To address this issue I propose to test whether sex differences exist in neurogenesis in the aging brain by examining proliferating cells and their progeny in the DG of young adult and aged mice, comparing male and females. The generation of neural cells will be assessd by (1) the uptake of bromodeoxyuridine (BrdU) in the DNA of dividing cells and immuno-histochemical detection of BrdU, and (2) immunofluorescent triple-labeling for BrdU and neuronal and glial markers and confocal microscopy to assess the phenoype of surviving BrdU-labeled cells. Absolute numbers of cells will be quantified using unbiased stereologic counting methods. Mice will receive BrdU injections and brains will be harvested 1 day later to determine sex-specific effects of age on the proliferation of cells, or 4 weeks later to examine sex differences on the survival and differentiation of the newly generated cells. To determine whether any sex differences in neurogenesis during aging are specific to the DG, I will also compare levels of neurogenesis in another neurogenic region, the subventricular zone of the lateral ventricular wall. This work will be involving the MCIC core training function to learn stereological counting methods. Results of this work will provide fundamental knowledge of how aging affects neural stem cells and their progeny Moreover, these results will provide baseline parameters for future studies aimed at determining whether neurogenesis in the aged brain responds to environmental factors that influence younger brains. Further, these parameters will provide a context to pursue the role of specific genes on neural precursor cells and their functional consequences in the aging brain. For example, genetic mechanisms underlying gender effects of aging neurogenic populations can be explored using transgenic knock-out and gain of function mutations. Moreover, the molecular basis for age/gender-related changes can be explored by comparing transcript profiles of neural precursor cells using DNA microchip arrays. Pilot 1, Year 3 "Insulin Signaling Pathway Genes and Aging in Nasonia"
Abstract Genes involved in the insulin-signaling pathway have been implicated in aging in organisms as diverse as nematodes, insects and mammals. However, little is known about the role of insulin-signaling genes in natural variation in aging among and between species. We propose to investigate differences in longevity and activity level between two closely related insect species in the genus Nasonia. Males of one species (N. vitripennis) develop more quickly, are more active, and live longer under caloric limitation than do males of the other species (N. giraulti). These differences can be studied genetically because the species are interfertile, allowing movement of genes between the species. Furthermore, we have partially sequenced and mapped 5-insulin signaliing genes from the two species (Pten, InR, Tor, Pi(3)k and S6k). Preliminary results indicate that at least one gene (Pten) has undergone rapid amino acid evolution since divergence of the two species. Our specific goals are to (1) determine whether insulin-signaling pathway genes are associated with differences in longevity and activity between the species, (2) use molecular evolutionary methods to determine whether coding or regulatory regions of insulin-signaling genes have undegone rapid evolution in the species complex and (3) use selection experiments to determine whether there are other major genes affecting longevity differences between the species. Insulin-signaling genes will be introgressed from N. giraulti into N. vitripennis and marker assisted recombinaiton will be used to determine whether longevity and activity differences are tightly associated with insulin-signaling genes. Selection experiments will reveal whether other major genes are involved in longevity and activity differences between the species. Although Nasonia is not a traditional model organism, it has several features suited for evolutionary genetic studies of aging. These include ease of handling, short generation time, interfertile species and abundance of visible and molecular markers. Outside of Drosophila, Nasonia is the only insect for which the insulin-signaling genes have been both partially sequenced and mapped. By taking advantage of the particular features of the Nasonia system, this study will provide among the first information on the genetic basis of interspecies differences in longevity, and will determine whether insulin-signaling genes are good candidates for the interspecific differences in aging and activity.
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