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2016 Research Showcase
IVCB Abstracts
Abstract Title : Exploring the Effects of Heterozygous Deletions of the BECN1 and MAP1LC3B Genes in Ovarian Cancer Cells
Abstract : Heterozygous deletions of autophagy genes BECN1 and MAP1LC3B were found in over 95% cases of serous ovarian cancer, suggesting a connection between ovarian cancer and autophagy. Our research project aims to study how heterozygous losses of these genes can affect autophagy. The CRISPR/Cas9 system will be used to create these deletions in ovarian cancer cells. We hypothesize that a heterozygous loss in autophagy genes will reduce autophagosome count, which we hypothesize that will increase sensitivity to common chemotherapy drugs and decrease sensitivity to most autophagy-inhibiting drugs.
Abstract Title : The efficacy of a TLR7 agonist conjugated to silica nanoshells as an immune adjuvant in innate immune cells
Abstract : The ultimate goal of cancer immunotherapy is the recognition and destruction of tumor cells by the immune system. Recently, agonists of the toll-like receptors (TLR) have been recognized for their anti-tumor potential across various therapeutic modalities. The 'danger signals' they provide augment the host immune system response against tumor antigens by enhancing the local immunogenicity of the tumor microenvironment. The proposed project aims to evaluate the efficacy of an immune adjuvant, a TLR7 agonist, conjugated to the surface of hollow silica nanoshells. It is theorized that addition of the TLR7 agonist when conjugated to a nanoparticle stimulates immune recognition of tumor antigens leading to a sustained systemic anti-tumor immunity. Preliminary in vitro studies in RAW cells, a macrophage cell line, have shown that the immunostimulatory properties of a TLR7 agonist are enhanced when conjugated to 100nm silica nanoshells. The nanoparticle is postulated to mimic the presentation of TLR7 agonists similar to antigen recognition on a virus.. Optimizing the conjugation synthesis will aid in effectively measuring how TLR7 density could affect dose response. Through addition of immune adjuvant, the extent of the induced immunogenic response will be investigated in RAW cells and bone marrow derived dendritic cells (DCs) and macrophages with the aim to enhance induction of anti-tumor cytokines.
Abstract Title : Sex Differences in Toll-Like Receptor Regulation of Ankle Swelling in the K/BxN Mouse Model
Abstract : Recently there has been an emphasis to evaluate the differences between male and female mice in animal models of human diseases. Rheumatoid arthritis is an autoimmune disease that occurs predominantly in females. In the K/BxN serum transfer model of murine arthritis, arthritis is usually robust in the recipients of the arthritogenic sera. However, the differences in males and females have not been carefully dissected in wild type and targeted mutant strains. Although autoimmune disease is mediated by the adaptive immune lymphocytes, innate immune mechanisms have also been implicated. Toll-like receptor (TLR) signaling is key to innate immune responses and TLR 3, 7 and 9 have been specifically implicated in modulating autoimmune diseases. Major ligands for TLR3, TLR7 and TLR9 are dsRNA, ssRNA and CpG DNA respectively. Here, we focused on mice deficient in these Toll-like receptors to evaluate the sex differences in ankle swelling after the induction of passive serum transferred arthritis. Wild type (WT) C57Bl/6 females had less swelling than Tlr3-/- mice on days 5 and 6 (P<0.001; by two way ANOVA with Bonferroni post hoc test) and Tlr9-/- females on days 8 and 9 (P<0.05) and more swelling than Tlr7-/- females on days 4-10 (P<0.001). WT male mice had more swelling than Tlr7-/- males on days 3-10 (P<0.001); however there were no differences in swelling compared to Tlr3-/- and Tlr9-/- males. WT and Tlr7-/- males developed swelling more rapidly than the female mice with significant differences on days 3 and 4 (P<0.001). However Tlr3-/- and Tlr9-/- mice did not exhibit any differences between male and female mice. In female mice TLR3 and 9 may have anti-inflammatory roles and TLR7 is proinflammatory. Only TLR7 played a role in the development of arthritis in male mice suggesting that the regulation of swelling is different between males and females and warrants further investigation.
Abstract : Recently, Hollow Silica Shells have been explored for various biomedical applications due to their low toxicity, high biocompatibility and synthesis advantages. Amongst the latter, is the tunability of the structural characteristics of the shells (such as diameter, wall thickness and pore size), as well as the ability to incorporate a variety of drug loading and doping methods into the synthesis. At present, we are using MRI imaging to characterize the mechanical ablation of tumors using High Intensity Focused Ultrasound (HIFU) with Hollow Silica Shells as a sensitizing agent to shift the ablative threshold towards a more controllable and safer range by avoiding thermal ablation and allowing real time imaging of the ablated region. For this purpose, we have prepared shells doped with Gd, in order to observe the area of ablation via MRI while applying HIFU. We have found that Hollow Silica Shells can act as a HIFU sensitizing agent, and have observed mechanical ablation occur, as the Gd released by shell breakage is visible under MRI. A further step is to develop particles that allow multimodal imaging (MRI, US) as well as ablative destruction of tumors, thus facilitating the non-invasive treatment of tumors.
Abstract Title : Modulation of Antiviral Signaling in Hepatitis B Virus Infection
Abstract : The Hepatitis B Virus (HBV) evades the immune system's response and suppresses interferon production. Previous reports have demonstrated that the mitochondrial antiviral signaling protein (MAVS) is inhibited by HBV expression. However, the precise mechanism behind this inhibition is still elusive. We hypothesized that the Parkin protein exerts some effects on MAVS signaling. Upon knocking down Parkin in HBV infected cells, we observed an increase in ISRE expression. We next determined that the HBx protein is responsible for upregulated Parkin expression and may cripple MAVS signaling. This suggests that HBx is involved in HBV's exploitation of Parkin, downregulating interferon production. In future experiments, the HBx defective genome of HBV will be used to explore the mechanism behind the MAVS-Parkin interplay. In addition, the MAVS signalosome will be examined to see how HBV or HBx disrupts MAVS's interaction with partner molecules like TRAF2, 3, 5 and 6 in HBV infected cells.
Abstract Title : Quantitative Method For Monitoring Murine Arthritis Development
Abstract : Rheumatoid Arthritis is an autoimmune disease that can lead to the chronic inflammation of synovial tissue resulting in bone, cartilage and joint deterioration. However, the cause of this disease is elusive and not entirely known. One important objective in studying any disease is to develop methods of monitoring the development and progression of its pathogenesis. In mouse models of rheumatoid arthritis the ankle thickness is measured with a caliper and the mice are typically monitored for paw swelling and erythema by visual inspection. Here we have developed a system of measurement to quantitate the swelling of individual toes increasing the objective data set per mouse. Mice were injected with K/BxN serum to induce arthritis on Day 0 and the ankle thickness was serially measured for 10 days to ensure the development of arthritis. The mice were positioned under sedation with Velcro tabs and the plantar aspect of the foot was photographed under white light with a fixed distance copy stand. The images were transferred to ImageJ and the outline of each toe was drawn and the area quantified. As the first toe is smaller than the others thus reducing its area, the toe areas were divided by their respective lengths to generate an aspect ratio. Based on these measurements, the profile of swelling could be tracked for each individual toe. This system was validated using a separate model of arthritis which is antigen induced, the collagen induced arthritis model.
Abstract Title : The Role of Id2 in Transcriptional Regulation of Memory CD8+ T cells
Abstract : CD8+ T cells are essential components of the immune system that provide protection against intracellular pathogens and tumors. It is important to understand the transcriptional networks that guide CD8+ T cells to differentiate and maintain heterogeneous effector and memory populations following infection. The E protein transcription factors and their inhibitors, Id proteins, play vital roles in T cell differentiation and maintenance. Previously, Id2 has been shown to regulate effector CD8+ T cell differentiation and survival following infection. Id2 expression is maintained in memory populations; therefore, we hypothesize Id2 also functions in CD8+ T cell memory formation, maintenance and function. We have established an inducible knockout model, which gives us the capability to control the temporal deletion of Id2 and will allow us to evaluate Id2 regulation of E proteins in memory T cell populations. Understanding the unique transcriptional programs that define CD8+ T cell memory will allow for improved vaccine design.
Advisor : DR. CLARK CHEN
Abstract Title : TMZ-induced microRNA degradation upregulates DNA repair in Glioblastoma
Abstract : Glioblastoma is the most common form of primary brain tumor. It is an aggressively infiltrative tumor that is intrinsically resistant to DNA damaging agents, including conventional chemotherapy and radiation therapy. Our lab previously demonstrated that this resistance is mediated by the microRNA miR-181d. Here we demonstrate the molecular mechanism by which miR-181d regulates key DNA repair processes, including homologous recombination (HR). Using unbiased profiling experiments and miRNA binding site prediction algorithms, we found key HR genes as downstream effectors of miR-181d. Exogenous expression of 181d suppresses HR capacity in glioblastoma. Moreover, induction of DNA damage triggers degradation of miR-181d, causing up-regulation of HR and resistance to chemotherapy and radiation therapy. Consistent with these observation, key HR genes such as Rad51, are significantly up-regulated in clinical glioblastoma specimens after chemotherapy. Our results indicated miR-181d is a master regulator miRNA that determines glioblastoma response to radiation therapy and chemotherapy.
Abstract Title : The non-apoptotic roles of caspase-8 in tumor cell malignancy
Abstract : Caspase 8 is a cysteine protease that plays a key role in apoptosis, a type of programmed cell death. However, and a bit unexpectedly, elevated levels of this protein are found in many cancer cell lines. We hypothesize that caspase-8 might also play other key roles in cells. We evaluated how caspase-8 plays a role of cell fitness in 4T07 mouse breast cancer cells. On tissue culture plastic, the expression of caspase-8 or knockdown of caspase-8 gave similar levels of cell survival. In 3-dimensional agar, caspase-8 was detrimental to clone formation. Despite this disadvantage in survival in 3D, cells that express caspase-8 were more highly metastatic in mice compared to cells with caspase-8 expression suppressed. Since caspase-8 has been linked to cell adhesion, and since cells in 2D have a more rigid substrate to adhere to, we tested the impact of caspase-8 on the adhesion of these 4T07 cells. I found that cells that express caspase-8 were not only more efficient at binding to extracellular matrix proteins, but were also able to activate calpain-2 better within their focal adhesions. Calpain-2 is known to facilitate cell adhesion, spreading and migration. Therefore, my results support a model in which tumor cells are able to re-purpose and may even come to depend on caspase-8.
Abstract Title : Host-Pathogen Interactions of Candida Albicans with Human Neutrophils
Abstract : Neutrophils are an essential component of the immune system and are a critical first line of defense against pathogens. The main function of neutrophils is to prevent infections by detection of pathogens and employment of multiple killing mechanisms. These mechanisms include generation of reactive oxygen species (ROS), release of antimicrobials, uptake of pathogens and release of neutrophil extracellular traps (NETs). Defective, impaired neutrophil responses render the host to become susceptible to infections. Common (fungal) infections in immune-compromised patients (i.e. genetic defects e.g. CDG or chemotherapy/surgery) are caused by Candida albicans. C. albicans is an opportunistic pathogen causing serious life-threatening infections. Our goal is to study the molecular mechanisms of this host-pathogen crosstalk. We found that healthy neutrophils are capable of efficiently killing C. albicans and observed that healthy neutrophils respond to C. albicans via production of ROS. When blocking this critical, antimicrobial mechanism from neutrophils using a pharmacological agent that phenotypically mimics neutrophils in CDG patients, we found an increased fungal burden demonstrating an important role for ROS production in resistance to C. albicans. In summary, our data suggest that ROS is important for clearance of C. albicans by human neutrophils and that strategies aiming to boost or re-activate ROS production might be a potential novel therapeutical target to boost the innate immune functions in immune-compromised patients to prevent C. albicans infections and/or to overcome the rise of resistance development in C. albicans against antifungals.
Abstract Title : The potential role of DNA damage and repair by NEIL2 in bacteria associated cancer
Abstract : Bacterial infection causes inflammation, increased production of reactive oxygen species (ROS) and subsequent oxidative DNA damage. These are all well-known risk factors in cancer. The two most common bacterial infection associated cancers are Helicobacter pylori mediated gastric carcinoma and fusobacterium associated colon carcinoma. However, the detailed molecular mechanism(s) of elevated DNA damage after infection that can trigger carcinoma is still unknown. Commonly, oxidized bases are repaired via the DNA base excision repair (BER) pathway, which is initiated with the excision of the lesion by DNA glycosylases. NEIL2, a major DNA glycosylase involved in base excision repair, is studied here in gastric and colonic epithelial cell lines following infection with H. pylori and fusobacterium respectively. NEIL2 level was decreased in gastric epithelial cells following infection with H. pylori in a dose and time dependent manner. The decreased level of NEIL2 after infection may play a significant role in elevated DNA damage in the epithelial cells that can increase the risk of cancer following infection. Characterization of the effect of bacterial infections on DNA repair proteins will provide greater understanding on bacteria associated cancers and will help in the recognition of early cancer biomarkers that may be targeted for cancer therapies and treatment.
Abstract Title : The Efficacy of 1V270 Immunoactivator Molecule in Inhibiting Breast Cancer Metastasis
Abstract : Cancer immunotherapy aims to boost the body's natural defenses to fight cancer. Through the use of materials naturally produced by the body or synthetically derived in a laboratory, cancer immunotherapy treatment methods can be made to help the immune system become more efficient in destroying cancer cells. In this project, we will use a mouse syngeneic model of human breast cancer, namely the 4T1 model. Its highly tumorigenic and invasive qualities allow for the spontaneous metastasis from the primary tumor in the mammary gland to other distant areas including lymph nodes, liver, blood, lungs, and bone. The Carson laboratory has developed toll-like receptor (TLR) 7 ligand IV270, a small innate immunoactivator molecule. Toll-like receptor 7, key components in innate and adaptive immunity, recognizes the ligands nucleoside structures and thus induces the response of proinflammatory cytokines, chemokines, and type I interferons (IFNs). Preliminary data indicates that systemic administration of TLR7 ligand, IV270, effectively inhibits breast cancer metastasis in lungs in 4T1 syngeneic model. With the obtained the knowledge that TLR7 ligand activates innate immune responses, it is hypothesized that innate immune cells in the lungs are involved in anti-metastatic effects by 1V270. Therefore the project aim is 1) to determine optimal treatment schedules and explore combination therapy that enhances antitumor effects by IV270 and 2) to determine responsible cells in lungs for inhibition of lung metastasis by 1V270.
Abstract Title : Sex differences in Toll-like receptor regulation of arthritic pain in mice
Abstract : Autoimmune diseases like rheumatoid arthritis are regulated by both adaptive and innate immune systems. We previously described the persistence of mechanical allodynia in male mice after the resolution of swelling in a passive serum transfer model of arthritis. This persistent allodynia had pharmocological features of neuropathic pain in that it was responsive to gabapentin, but not non-steroidal anti-inflammatories or anti-TNF. We found that Toll-like receptor 4 regulated the onset of this allodynia. In my project, I aimed to evaluate the role of other Toll-like receptors in arthritic pain and the differences of pain and swelling between male and female mice. Using the K/BxN serum transfer model of murine arthritis, I tested a series of mice that were deficient in single or multiple Toll-like receptors and their signaling pathways. Male and female mice were tested and compared. Wild-type female mice can resolve their pain. However, other strains of Toll-like deficient mice cannot resolve their pain in the post-inflammatory phase.
Abstract Title : The Effect of murine Schlafen proteins on retroviruses such as HIV
Abstract : Human Schlafen protein 11 has been proved to inhibit HIV, human immunodeficiency virus, in a codon-usage-based manner. The purpose of this project is to compare the anti-retroviral effect of murine Schlafen proteins to that of human Schlafen 11. Eight murine Schlafen DNAs which are mSlfn1, mSlfn2, mSlfn3, mSlfn5, mSlfn8, a shorter analog of mSlfn8, mSlfn9 and mSlfn10, along with HIV (pnl43) are transfected into 293T cells and harvested for western blot and qPCR. Human Schlafen 11 is used as positive control whereas wildtype GFP serves as the negative control. Western blot is probed for p24, which is a cleaved product of p55, a tag for HIV. The absence of bands near 24kDa on western blot would prove the inhibition on HIV by the murine Schlafen, and the level of mRNA quantified by qPCR would show whether the inhibition of HIV occurred prior to or after transcription.