2001 — 2005 |
Leaman, Douglas W |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Molecular Mediators of Interferon Induced Apoptosis
DESCRIPTION (Provided by the Applicant): Interferons (IFNs) are multifunctional cytokines that have proved effective in treating hematologic and solid tumors. Regulation of gene expression within tumor cells by IFNs results in the antitumor effects for most tumor types. Antiproliferative and immunomodulatory effects have received the greatest focus as underlying cellular mechanisms of antitumor action of IFNs. However, cell loss contributes to antiproliferative effects of IFNs in the majority of tumor cell lines. In comparing the effects of IFN-B to those of IFN-a2, IFN-B was found to induce apoptosis in a broad range of tumor cell types. To identify genes that mediated IFN-dependent apoptosis, oligonucleotide gene array studies were performed using pooled RNA samples from sensitive WM-9 melanoma cells left untreated or treated with IFN-alpha-2 or IFN-beta for 8, 18 and 40 h. Those studies identified novel induced genes whose protein products are known, or hypothesized to regulate apoptotic processes including TNF-Related Apoptosis Inducing Ligand (TRAIL) and XIAP-associated factor-1 (XAF-1). Expression of these genes was correlated with IFN-beta induced apoptosis in sensitive, but not resistant cells. Studies aimed at affirming a functional role these proteins in mediating IFN-beta-dependent apoptotic responses are proposed. These include studies designed to neutralize TRAIL or XAF- 1 function in sensitive cells, and studies aimed at restoring their expression in unresponsive cells (Specific aims land 2). Although the phenomenon of IFN-beta-specific gene induction has been described in the literature, the mechanisms involved are poorly understood. The TRAIL gene will therefore be used as a tool to probe mechanisms of differential gene regulation by IFN-beta as compared to IFN-alpha (Specific aim 3). The results of these studies should provide important information on the molecular details of antitumor activity of IFNs that can be used to identify or develop targeted therapeutics that augment IFN effects.
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0.958 |
2006 — 2010 |
Leaman, Douglas W |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Impact of the Interferon Regulated Proteins Xaf1 and Znf313 On Innate Immunity
DESCRIPTION (provided by applicant): To control viral infections the host has developed an integrated defense network that comprises both innate and adaptive immune responses. Initial responses to viral infection involve primarily the innate response and the killing of infected cells with cytotoxicity. Once the virus has invaded the cell, host-mediated responses are triggered by single stranded and doubled stranded viral RNAs that induce expression of stress cytokines including interferons (IFNs). IFNs are essential for initiating and coordinating a successful antiviral response by inducing a number of intracellular genes, the IFN-stimulated genes (ISGs), which prevent virus replication/cytolysis and stimulate the adaptive arm of the immune system. Cellular mechanisms that prevent viral replication, dissemination or persistent infections include global inhibition of protein synthesis, blockage of protein transport and induction of apoptosis. A number of cellular proteins have been implicated as mediators of virus-induced apoptosis, including two of the proteins that were the focus of our original proposal, TRAIL/Apo-2L and X-linked inhibitor of apoptosis-associated factor-1 (XAF1). IFNs also potently regulate cellular responsiveness to virus and other cellular pathogens by upregulating components of the virus detection system, including toll-like receptors, RIG-I and downstream signaling molecules. Recently, we have shown that XAF1 augments cellular sensitivity to apoptotic agents such as TRAIL, and can also enhance cellular antiviral responses. XAF1 is shown herein to impact signaling cascades initiated by TNF family ligands and dsRNA signaling cascades. This grant is designed to functionally dissect the mechanism of XAF1 activity. Emphasis will be on downstream regulators of XAF1 activity, including a newly identified protein, ZNF313, which we now show to be a XAF1-binding RING finger protein that is post-translationally regulated by IFNs. The role of this putative E3 ubiquitin ligase in regulating IFN, viral and other innate immune responses will be pursued as outlined in the following aims. We will also pursue recent data linking XAF1 to other signaling molecules that regulate ubiquitin ligation, including TRAF2 and RIP2, and demonstrate that TRIP6, another XAF1 binding partner, may represent a novel ubiquitination regulator in the TNF/dsRNA pathway.
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0.958 |
2008 — 2009 |
Ashburner, Brian [⬀] Leaman, Douglas Chadee, Deborah (co-PI) [⬀] Shemshedini, Lirim (co-PI) [⬀] |
N/AActivity Code Description: No activity code was retrieved: click on the grant title for more information |
Mri: Acquisition of Instrumentation For Digital Imaging and Fluorescent Detection
A grant has been awarded to the Department of Biological Sciences at The University of Toledo to support the acquisition of: 1) the Storm 860 Gel and Blot Imaging system; 2) the ImageQuant RT ECL Imaging system; and 3) a SpectraMax M5 Multi-detection plate reader. This instrumentation will support the research programs of the PI and Co-PIs and other members of the Department of Biological Sciences focused on cell signaling and regulation of gene expression in human cells and in model systems (nematodes and plants). In addition, these instruments will also be available to the broader research community in other departments at The University of Toledo. These instruments will provide investigators with more advanced and sensitive tools for conducting assays that they currently do not have the capabilities of performing.
The acquisition of these instruments will have a significant broader impact on the educational mission of the Department of Biological Sciences and The University of Toledo. The PI/Co-PIs as well as the department as a whole are strongly committed to the integration of research and education. The use of these instruments will be available to undergraduate students working on research projects under the direction of a faculty mentor. In addition, the use of these instruments will be incorporated into several upper division undergraduate laboratory courses, in particular the core Molecular Genetics and Cell Biology (taught by the PI) labs. Underrepresented groups, through the department?s commitment to the STARS and Glenn-Stokes programs will also benefit from the acquisition of these instruments.
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1 |
2009 — 2010 |
Leaman, Douglas W |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
Role of Isg12 in Cellular Innate Immune Responses
DESCRIPTION (provided by applicant): Interferons (IFNs) are multifunctional cytokines that play a critical role in innate immunity. Type I IFNs (IFNs-1, -2, -I and others) exhibit immunomodulatory, antiproliferative and antiviral activities, although gene knockout experiments have suggested that, among these effects, the antiviral function is most critical for survival. IFNs elicit their effects through the induction of discreet subsets of early response genes, known collectively as IFN-stimulated genes (ISGs). To date, however, the precise functions of many individual ISGs remain poorly understood. We have performed extensive gene expression profiling of genes regulated by type I IFNs in a variety of normal and malignant human cell types. These studies have highlighted large repertoires of both common (e.g. induced in all cell types) and unique (cell-specific) ISGs. Here, we propose to assess the physiological function of the protein encoded by the ISG12a gene, an IFN-induced gene that was implicated in our gene array studies as an ISG strongly induced in all cell types. Our recent studies have shown that the ISG12a protein is localized to the mitochondrial membrane, and that ectopic ISG12a expression results in a sensitization of cells to other apoptotic stimuli. Since apoptotic responses to virus are an important component of an overall IFN response, we hypothesize that ISG12a influences the ability of cells to respond to viral infections by impacting mitochondrial function, and possibly regulating apoptotic responses. Thus, we propose to evaluate the physiological importance of endogenous ISG12a in both IFN-dependent mitochondrial responses and in antiviral effects by conducting exploratory studies that encompass these specific aims: Aim 1. To determine the significance of ISG12a in IFN-induced cellular and mitochondrial changes Aim 2. To determine the significance of ISG12a induction on antiviral and other innate immune responses PUBLIC HEALTH RELEVANCE: The ability of the body to respond to invading organisms, including virus and bacteria, depends on the coordinated actions of both immune cells and other cell types in the body. Production of interferon by virally infected cells upregulates the body's ability to sense and respond not only to other viruses, but also to other invading microorganisms. We have identified an interferon-induced protein, called ISG12a, that we feel is involved in this sensitization process, and better understanding of ISG12a's effects on cells should lead to improved understanding of how the body regulates responses to virus and other pathogens.
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0.958 |
2014 — 2018 |
Leaman, Douglas Stepien, Carol |
N/AActivity Code Description: No activity code was retrieved: click on the grant title for more information |
Collaborative Research: Gene Diversity of the Vhs Fish Virus: Evolution of Cellular Immune Response and Pathogenesis
Viral Hemorrhagic Septicemia virus (VHSv) is one of the world's most important finfish diseases, impacting both sport fishing and commercial fishing ventures around the globe. VHSv emerged only recently in the Great Lakes, and little is known about how it arrived and why it periodically leads to large scale fish die-offs. The proposed project will examine how VHSv's evolution in the Great Lake region has contributed to its ability to cause disease, and should aid informed predictions about future outbreaks. Thus, the investigations will integrate studies on VHSv evolution with identification of the means by which the virus disrupts immune systems of infected fish, leading to sickness or death. These results should help to identify how specific genetic changes in the virus can lead to either enhanced or reduced impact on infected fish, and thus will provide critical data for understanding the connection between viral genetic changes (i.e., viral evolution) and the host's response. Results will contribute towards more effective prevention, prediction, and control of VHSv, aiding national and international management efforts. Although the work will focus on the spread of VHSv within the Great Lakes region, the studies will have application to other viruses (fish and otherwise). The PIs will design an attractive mobile display for museum exhibits and broader distribution at conferences and public meetings to assist in disseminating the information beyond the scientific community, and will train both graduate and undergraduate students as part of this project.
Each of the aims of the project addresses a specific question about the spread of this disease in the Great Lakes region. Evolutionary diversification patterns and rates of VHSv genes will be evaluated using each protein gene (nv, N, M, L, and P) in strains and substrains, using new and existing samples. Results will determine how these are related to viral selection in new outbreak taxa and areas, and whether and how strains become endemic or epidemic. Cellular and host responses will be assessed to determine how mutations within viral genes that repress fish innate immune responses affect function by comparing VHSv M and P (and other) gene functions among VHSv strains. The project leaders will use reverse genetics to assess the role of M, P and L in modulating innate immune responses and viral pathogenicity. Taken together these results will be valuable in the present understanding of a new emerging and challenging infectious disease.
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1 |
2019 — 2020 |
Leaman, Douglas W |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Prevention of Adenovirus Pathogenesis Through Downregulation of the Apical Adenovirus Receptor @ Wright State University
PROJECT SUMMARY/ABSTRACT Adenoviruses (AdV) are common human pathogens that cause typical cold symptoms in healthy indi- viduals, but can potentially progress to acute respiratory distress syndrome (ARDS) with up to 50% mortality, particularly in highly susceptible, immunosuppressed people, and new, potentially lethal variants continue to emerge each year. No therapeutic that specifically prevents or treats AdV infection exists and the development of novel treatments would prevent the morbidity and potentially mortality associated with AdV infection. The objective of this proposal is to determine the mechanism of action and anti-adenovirus efficacy of novel molecules that decrease apical Coxsackievirus and adenovirus receptor (CAREx8) protein expression. Our central hypothesis is that decoy peptides that block the interaction between MAGI-1 and CAREx8 destabi- lize apical CAREx8 protein in the airway to abrogate AdV entry and pathogenesis. We will test our hypothesis using polarized model epithelia with Dox-inducible CAREx8 expression, well-differentiated primary human and rodent airway epithelia to validate our findings, and the cotton rat model to evaluate wildtype AdV pathogenesis in immunocompetent and cyclophosphamide-immunosuppressed animals, with two specific aims: Aim 1: To elucidate the mechanism of MAGI-1 activating peptide and AdV triggered proteolytic degra- dation of CAREx8. Completion of this aim will allow the identification and development of novel and much needed targets and approaches to prevent the infection and spread of pathogenic wild-type AdV and, in the future, group B coxsackieviruses. Aim 2: To define the protection afforded by MAGI-1 PDZ1 binding peptides against wild type adenovi- rus infection in cotton rats. Completion of this aim will provide proof-of-principle that AdV infection can be thwarted by reducing its primary receptor and may save lives of severely infected or immunosuppressed indi- viduals. Overall impact: Understanding the mechanisms that regulate the expression and localization of CAREx8, and how this unique isoform mediates viral entry at the apical surface of a polarized epithelium is criti- cal for understanding viral spread, tissue tropism, and pathogenesis. The successful completion of the pro- posed aims will identify not only the cellular mechanisms regulating the expression of the apical adenovirus receptor but also mechanisms regulating viral binding and infection. This will establish the feasibility of thera- peutic agents that reduce the susceptibility of the airway epithelium to adenovirus infection and pathogenicity prior to infection and during an active infection in immunocompetent and immunosuppressed conditions. We ultimately expect the proposed aims to lead to novel anti-viral interventions that may also block other viruses that use CAR as a primary receptor, and provide insight into the regulation of other related viral receptors.
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0.904 |