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2015 Research Showcase
IVCB Abstracts
Abstract Title : Chromatin Immunoprecipitation of H3K27Me3 and H3k4Me3 in WT and Ezh2-/- Murine Neutrophil Progenitors During Differentiation
Abstract : Myelodysplastic syndromes (MDS) are clonal bone marrow disorders characterized by blood cytopenias and potential progression to acute myeloid leukemia (AML). A small portion of MDS cases with poor prognosis are characterized by genetic aberrations in EZH2, the catalytic subunit of polycomb repressive complex 2 (PRC2) which trimethylates lysine 27 of histone H3 (H3K27), a histone modification associated with gene repression. HoxB8-ER cells are murine myeloid progenitor cells which can be differentiated in vitro into neutrophils. The absence of Ezh2 in these cells (Ezh2-/-) was previously shown to produce aberrant neutrophils after a six day differentiation period, with abnormal migration, phagocytosis, and overproduction of reactive oxidative species. To understand how Ezh2 loss mediates these phenotypes, chromatin immunoprecipitation and sequencing (ChIP-seq) was used to study the genetic loci with altered trimethylation of histone H3. Furthermore, lysine 4 (H3K4) trimethylation is inversely related to the trimethylation of H3K27. ChIP-seq will be used to verify this correlation and determine which genes are marked with respect to H3K27 and H3K4 in Ezh2-/- and control cells. For both measurements, ChIP was performed over the course of cell differentiation at four evenly placed time points and the libraries were subsequently created for sequencing. Ultimately, the study aims to ascertain which genes are dysregulated by mutations in EZH2 in order to identify potential therapeutic targets for individuals with MDS.
Abstract Title : Enhancement of Potency of a TLR7 Ligand by Conjugation to Polysaccharides
Abstract : Synthetic small molecule Toll like receptor 7 (TLR7) ligands are effective topical agents for anti-viral and anti-tumor therapy by activation of the innate immune response in the host. However, currently available small molecule synthetic TLR7 ligands cannot be administrated systemically due to poor solubility, lack of specificity and short pharmacokinetic half-life. Here, we hypothesized that conjugation of a low molecular weight TLR7 ligand to polysaccharide would enhance its potency by increasing cellular uptake efficiency, and improve pharmacokinetics in vivo. The synthetic TLR7 ligand, 2-methoxyethoxy -8-oxo-9-(4-carboxy benzyl)-adenine (1V209), was covalently conjugated to Ficoll or dextran. These conjugates were highly water soluble, stable for at least 5 months at room temperature in aqueous solution, and easy to lyophilize and reconstitute without altering the potencies. The conjugates showed significantly higher potencies than unconjugated drug both in vitro and in vivo. When the conjugates were used as a vaccine adjuvant, they enhanced antigen specific humoral and cellular immune response to a greater extent compared to unconjugated TLR7 ligands. These results indicated that small molecule TLR7 ligands conjugated to polysaccharides significantly improve immunostimulatory potency.
Abstract Title : Investigation of EPCR Regulation of Dendritic Epidermal γδ T Cell Rounding
Abstract : Dendritic Epidermal T Cells (DETC) are dendritic γδ T cells that are resident in the epidermis. In unwounded epidermis, DETCs are in contact with neighboring keratinocytes, monitoring for signs of stress or disturbances. In instances of injury, keratinocytes express a stress-‐induced self ligand that bind to DETC TCRs. It has previously been found that upon receiving stress signals, DETCS change their morphology by retracting their dendrites to take a rounded form. Endothelial Protein C Receptor (EPCR), expressed on keratinocytes, is an antigen that plays a critical role in regulating the activation of γδ T Cells. Though EPCR has been identified, little is known regarding the molecules that interact with EPCR to produce a signal to DETCs. Here, we investigate EPCRlow mutants in order to determine the impact of EPCR signaling pathway on DETC activation. By identifying and understanding the molecules that regulate γδ T Cells activation, chronic non-healing wound treatments and therapies may be improved upon.
Abstract Title : Survival Rate and Time of Death After Being Diagnosed with Medulloblastoma is Dependent on the Gene Amplification of MYCN, ALL3, and ODZ3 Genes.
Abstract : Medulloblastoma is a fast-growing malignant brain tumor with varying results upon a person's life: many may not live through it while others may survive until old age. Using the collection of publicly accessible data, data on tens of thousands of humans who once experienced medulloblastoma have been recorded based on the age of the person when diagnosed, what stage the the tumor was in, how long the person survived (if the person survived), along with the very type of gene amplifications these people had. Using these data, boolean plots can be made and analyzed to decide which gene amplifications or gene non-amplifications will affect a person with medulloblastoma, be it living longer or living at all. It is known that the V-Myc Avian Myelocytomatosis Viral Oncogene (MYCN) gene amplification results in a quicker mortality for a person with medulloblastoma, while a person without MYCN amplification is very strongly correlated to living much longer, and even surviving the medulloblastoma. The MLL3 and ODZ3 genes are analyzed with similar conclusions. It is hoped that in the future a doctor can test a person's genes and accurately tell how long the person has to live with medulloblastoma.
Abstract Title : Characterization of mTOR Pathway in Bladder Cancer
Abstract : Each year there are approximately 74,000 new cases of bladder cancer with 15,000 deaths, making it the 5th most prevalent cancer in the United States. Similar to other types of cancers, bladder cancer has been linked to dysregulation of the PI3K/Akt/mTOR pathway; a pathway which regulates cell proliferation, cell survival, and cytoskeletal reorganization. To find a potential therapeutic treatment for bladder cancer we tested the efficacy of mTOR inhibitor drugs, Torin-2 and KU-0063794, on inhibiting cell viability and migration in UMUC3, T24, and J82 bladder cancer cells. Immunoblot analysis revealed that both drugs inhibit PI3K/AKT/mTOR activity in a dose- and time-dependent manner. Further, Torin-2 (IC50 ~ 25nM) was found to be about 55 times more potent than KU-0063794 (IC50 ~ 1.4M) in inhibiting cell viability, and significantly inhibited cell migration. We conclude that mTOR inhibition using Torin-2 or KU-0063794 may potentially be useful for the therapeutic treatment of bladder cancer.
Abstract Title : Oncogenic Isoforms of the RON RTK Gene Are Constitutively Activated And Display Altered Signal Transduction Pathways
Abstract : The RON gene is a receptor tyrosine kinase that is often overexpressed in human pancreatic cancer and other epithelial cell types of cancers. While mutations in RON are rare, alternative splice variants of RON have been associated with the progression of epithelial cell types of cancers. Among these variants are two RON isoforms of particular interest: (1) Partial splicing of exons 5 and 6 (P5P6) and, (2) short form Ron (sfRON). P5P6 is a novel RON isoform previously unidentified; and sfRON is an isoform identified in a number of human and mouse epithelial cell cancers. P5P6 and sfRON proteins were immunoprecipitated to determine the tyrosine phosphorylation status of the proteins and both isoforms were activated as judged by reactions with phosphotyrosine antibody. The P13K-AKT and MAPK pathways are known to be activated by RON signaling; and immortalized human pancreatic duct cell lines expressing wild type RON (HPDE6) or P5P6 (HPDE6-P5P6) were examined by western blot to determine if P5P6 is activates these pathways . There was no difference in the activation of the P13K-AKT pathway comparing parental to P5P6 expressing HPDE cells as the pAKT pathway is constitutively phosphorylated in both cell lines. In contrast, we observed that the MAPK pathway was constitutively activated in P5P6 cells but not in parental HPDE cells. Lastly, parental, P5P6 and sfRON expressing HPDE cells were orthotopically injected into immune-compromised mice. Parental HPDE cells did not form tumors but the P5P6 and sfRON expressing cells grew tumors demonstrating that both isoforms are tumorigenic. These studies showed that a novel isoform of RON (P5P6) recently identified in our laboratory is constitutively active, activates the MAPK pathway and is tumorigenic in immune-compromised mice.
Abstract Title : Nicotine Signaling in Caco-2 Cells to Define Biochemical Readouts
Abstract : CHRFAM7A is a recently recognized CHRNA7 gene that encodes a subunit of the α7-nicotinic acetylcholine receptor (α7nAChR) and alters its ability to bind and respond to nicotine. Originally arising in the human genome by translocation and partial duplication of the CHRNA7 gene when humans diverged from great apes, CHRFAM7A is unique to the human-genome. Recently, we have shown that the CHRFAM7A gene is expressed in human leukocytes and gut epithelium, but its functions in these peripheral tissues are not known. For example, it may regulate α7nAChR activity and as such, the human inflammatory response to nicotine in both leukocytes and gut epithelium. Because both α7nAChR and CHRFAM7A are detected in gut epithelial Caco-2 cells, we investigated changes in intracellular signaling in Caco-2 cells after nicotine stimulation. To this end, Caco-2 cells were stimulated with nicotine and changes in the phosphorylation of ERK1/2 (pERK 1/2) were measured by immunoblotting using antibodies to pERK1/2. A dose and time course analyses with serum-starvation assays of Caco-2 cells established the experimental conditions that will allow us to study the effects of CHRFAM7A on intracellular signaling when it is over expressed in Caco-2 cells.
Abstract Title : Molecular dissection of WNT signaling in the PCSD new series of patient-derived xenograft models of bone metastatic prostate cancer. [DANIELLE BURNER: co-presenter]
Abstract : One of the leading health problems affecting men in the United States and many other parts of the world is prostate cancer. Cancer patients are surviving longer due to the prevalence of prostate-specific antigen testing for the detection of prostate cancer and improved therapies. However, over 80% of advanced patients develop bone metastatic cancer. Recently, the Jamieson lab revealed that there is something unique in the bone microenvironment and androgen deprivation alone is not sufficient to treat bone metastatic prostate cancer. Therefore, this project is focused on determining whether WNT5A, BMP-6, and WNT5A receptors including ROR1 and ROR2 components help prostate cancer become more aggressive and thrive in the bone microenvironment. These components and their downstream effectors regulate various processes that are important for cancer progression, including tumor initiation, tumor growth, cell death, differentiation, and metastasis. To determine whether these components were expressed at the RNA level in our lab?s new prostate cancer xenograft models, reverse transcriptase polymerase chain reactions have been performed. The results of this study showed that BMP-6 and ROR1 components are expressed in our prostate cancer xenograft models, as well as possible WNT5A expression in PCSD1 and PCSD5 but not PCSD13. Further testing is needed to confirm the WNT5A expression differences in the different bone metastases. Future experiments will utilize immunohistochemistry techniques to elucidate where these components are expressed in the sections.
Abstract Title : Ebola Prevention and Health Care Projects in Kpando, Ghana - A Cultural Perspective
Abstract : Over the summer of 2014, I partook in a global health assignment in Kpando, Ghana, collaborating with a local non-governmental organization, UNiTED (Unifying Neighbors Through Education and Development). My aim was to understand methodologies of health outreach and medical approaches in relation to the Ghanaian culture, contextualizing both global and international health from the Western perspective. During this time, the largest Ebola epidemic in history affected neighboring countries in West Africa, killing thousands. The virus was first discovered and identified in 1976; consequences of infection have been deadly. The recent outbreak has resulted in a fatality rate of up to 71%. In an effort to bring awareness to local community members about the virus and disease in case of a local outbreak, pamphlets, presentations, and outreach efforts were administered. Collaboration with local translators and residents were coordinated to produce an appropriate pamphlet and presentation, using acceptable language and method of communication to be best suited for the local population. The local St. Patrick Hospital was one of the venues where daily educational discussions were conducted. Patients were given the opportunity to learn, ask questions, and familiarize themselves with the outbreak, under the coordination of a staff health educator and myself. Additionally, I worked with staff and physicians. We discussed the situation of the outbreak, and educated myself of their outlook and attitude. Further, and importantly, I learned about diagnosing malaria and related diseases, and also took note of the methodologies followed for prescribing certain malarial medications to pediatric, adult, and elderly patients. A group of six patients (male and female, 16 to 64 years old) living in a nearby village outside of Kpando, from, suffered from chronic diabetes and hypertension, resulting in acute ulcers. In developing my assessment of local diseases, daily rounds were made with a local translator, to change dressings, record vital signs, offer support, and advise on seeking further care at Margaret Marquart Catholic hospital. There was a need to also assist another local woman who was physically disabled from medical condition. Daily rehabilitation exercises were performed, while also offering social support. Further, along with another volunteer, I traveled to an island in Lake Volta to understand the needs of local residents, and helped with maintaining a newly assembled clinic. In a community with no running water, electricity, or local physicians, learning to effectively run a clinic was challenging, yet insightful. Donations of dressings, medications, and instruments from volunteers were used, while training a local resident how to successfully deliver health care. Many techniques came from the text, Where There is No Doctor. My experience in Ghana was contextualized from a global and international perspective coming from a Westerner, while learning about and understanding various health care methodologies. The importance of listening to the community, patients, and understanding cultural values and perspectives has been shown to be necessary, to best suit the needs of individuals and the local population. Whether serving a local community or going abroad, it is essential to aim for personal cultural humility and local cultural competency when attending to health care needs.
Abstract Title : TLR4 signaling contributions to arthritic swelling and pain in the K/BxN mouse model of arthritis
Abstract : Patients with rheumatoid arthritis experience persistent pain despite clinical remission of joint swelling. Arthritis can be modeled in mice with passive transfer of K/BxN serum, which causes joint inflammation. This response is modulated by the TLR4 pathway which utilizes MyD88 and TRIF adaptors that result in pro-inflammatory cytokine and type-1 interferon release, respectively. We examined the contributions of these two pathways using Myd88-/-, Il1r1-/-, Tram-/-, Triflps2, Irf3-/-, and Ifnar1-/- mice in the K/BxN serum transfer model of arthritis. Three patterns emerged: no joint swelling and no pain [Myd88-/- and Il1r1-/-]; joint swelling which resolved, but pain persisted [wild-type, Tram-/-]; and significant swelling but only mild pain [Ifnar1-/- and Irf3-/-]. Immunoblots of wild-type spinal cord extracts showed decreased ERK/JNK signaling on day 3 and increased STAT signaling on day 6 of arthritis. These data suggest a role of MyD88/MAPK signaling in early pain and TRIF/STAT signaling in late pain.
Advisor : WEG M. ONGKEKO
Abstract Title : Alcohol-Dysregulated miR-30a and miR-934 in the Pathogenesis of Head and Neck Squamous Cell Carcinoma
Abstract : Head and neck squamous cell carcinoma (HNSCC) is the sixth leading cancer worldwide, accounting for nearly 900,000 newly diagnosed cases each year. Alcohol consumption is a well-established risk factor for HNSCC; however, the molecular mechanisms by which this promotes HNSCC pathogenesis and progression remain elusive. Through RNA-sequencing analysis, we identified 8 microRNAs (miRNAs) significantly upregulated in HNSCC patients with a documented history of alcohol consumption. We then verified these dysregulations in-vitro and determined that miR-30a and miR-934 were the most highly upregulated. Further investigation revealed that overexpression of these miRNAs resulted in increased cell proliferation and induced expression of the stem-cell genes CD44 and BMI-1. Furthermore, inhibition of these miRNAs resulted in increased expression of cancer-related genes, decreased sensitivity to Cisplatin, diminished expression of stem-cell genes, and reduced matrigel invasion. Alcohol-associated dysregulation these miRNAs may play a critical role in the mechanisms that govern the pathogenesis and progression of HNSCC.
Abstract Title : Group A Streptococcus survival in macrophages results in cell death and inflammation
Abstract : The obligate human pathogen group A Streptococcus (GAS) is the cause of "strep throat" as well as severe, life-threatening infections including necrotizing fasciitis ("flesh-eating disease"). One GAS factor contributing to disease is SpeB, a secreted protease that inactivates numerous host immune molecules. However, clinical isolates from individuals with severe invasive infection show selection for bacteria with mutations in CovRS, a regulator of SpeB. We examined if SpeB would affect survival within phagocytes by infecting macrophages with wild-type SpeB mutant or SpeB-complemented GAS. We monitored bacterial viability within macrophages by plating colony-forming units. Concurrently, we measured cell death by Lactate Dehydrogenase release and the release of inflammatory cytokines with reporter cells. Wild-type GAS survived intracellularly approximately twice as well as SpeB-mutant GAS. However, these macrophages also died more rapidly and released 150% more IL-1b. SpeB was found to be necessary and sufficient for the increased IL-1b release, which occurred independent of the Caspase-1 pathway that typically regulates its release. Therefore, expression of SpeB may pose a dilemma for the bacteria during infection- promoting intracellular survival may amplify the inflammatory response toward the pathogen.
Advisor : MARY CORR
Abstract Title : MD2 as a Regulator of Arthritis Pain in a Murine Model
Abstract : Pain is one of the most disabling symptoms in arthritis. Rheumatoid arthritis affects millions of Americans each year. The K/BxN serum transfer model of murine arthritis recapitulates some but not all of the features of human disease. In this model, the serum of arthritic mice confers arthritis in the recipient. In a prior collaboration with the Yaksh laboratory, we found that Toll-like receptor 4 (TLR4) was associated with the persistence of mechanical pain in mice after the resolution of acute paw inflammation. MD2 is a co-receptor for TLR4 and TLR2 that is soluble and does not transmit signaling. In this project, we aimed to determine if the profile seen in the TLR4 deficient mice would be amplified in the MD2 null mice as these mice would function as double deficient in both TLR4 and TLR2. MD2 deficient mice were given K/BxN serum and monitored for several weeks for paw swelling and pain. The MD2 mice developed arthritis similar to wild type mice, but had a significant delay in the onset of pain. In the TLR4 mice, only the resolution phase was affected, indicating that TLR2 and TLR4 in concert influence the onset of pain and then regulate the persistence of pain.
Abstract Title : Cell-Free DNA is an Endogenous Mediator in Inflammatory Arthritis
Abstract : Neutrophils expunge their nuclear DNA as a protective mechanism to ensnare invading microbes. These neutrophil extracellular traps have been implicated as an antigenic reservoir for autoimmune diseases such as lupus and vasculitis, which have autoantibodies to dsDNA and myeloperoxidase respectively. The role of NETosis in the pathogenesis of rheumatoid arthritis has yet to be defined; however, the essential activity of peptidylarginine deiminase type IV (PAD4) in NETosis and in citrullinating key antigens in RA potentially links these two. In a neutrophil-dependent murine model of arthritis, induction of arthritis was associated with increased levels of circulating DNA. PAD4 deficiency did not ameliorate arthritis, although NETosis was prevented; however, PAD4 deficiency in combination with caspase-1 deficiency dramatically reduced arthritis. Additionally, the severity of serum-transfer induced arthritis was refractory to DNase treatment in C57BL/6 mice but was significantly attenuated in caspase-1 deficient mice. In this model, the presence of PAD4 and NETs did not initiate arthritis, but might play a role in a self-perpetuating cascade reliant on the inflammatory activity of caspase-1.