Our students are required to complete a thesis as a culmination of their studies in the College of Biomedical Sciences. The thesis can be either a traditional laboratory-based project or a Capstone project. The substantial research project that is submitted shows the development of the student’s scientific approach to problem solving and critical thinking.
|Monday April 17th, 2017 Location: Jefferson Alumni Hall Rm. 307|
|1:00pm - 4:00pm|
|Tuesday, April 18th, 2017 Location: Jefferson Alumni Hall Rm. 407|
|9:00am - 11:00am|
|Wednesday, April 19th, 2017 Location: Jefferson Alumni Hall M-61|
|2:00pm - 3:30pm|
|Monday April 24th, 2017 Location: Jefferson Alumni Hall Rm. M-61|
MS Program Abstracts
ESTABLISHING A HUMAN GUCY2C TRANSGENIC MOUSE SYSTEM FOR GUCY2C-DIRECTED IMMUNOTHERAPY SAFETY EVALUATION.
AJ FLOWERS1, AE SNOOK1
1Department of Pharmacology and Experimental Therapeutics, Thomas Jefferson University, Philadelphia PA.
Colorectal cancer (CRC) is a lethal disease. Current treatments only extend life about twenty-four months, calling for a new therapeutic paradigm for the treatment of CRC. One avenue is adoptive T cell therapy, in which genetically modified T cells, with chimeric antigen receptors (CAR) are transfused into patients. Guanylyl Cyclase C (GUCY2C) has been identified as a cancer mucosa antigen (CMA) which would be a direct target of these CAR T cells. We have demonstrated that our CAR-T cells are highly effective therapies against human tumors in animal models, but lack adequate models to test their safety. Therefore, we attempted to establish a transgenic mouse model, with spatial and temporal control of human GUCY2C (hGUCY2C) expression to evaluate our immunotherapy. We found that the full transgene incorporated into the genome and that the transgenic DNA is successfully undergoing recombination in our targeted cells. However, we were unable to detect hGUCY2C expression by various assays (i.e. FACS, immunoblotting, immunofluorescence). Further analysis revealed that hGUCY2C expression was failing at the transcription level: no mRNA signal was detected in our mice using highly-sensitive qRT-PCR. Future studies will require establishment of new transgenic lines or identification of alternative model system to explore GUCY2C immunotherapy safety.
CHEMOTAXIS OF MURINE CYTOMEGALOVIRUS SPECIFIC CD8+ T CELLS TO THE SUBMANDIBULAR GLAND
T Furmanak1 , CM Snyder2
1Department of Microbiology and Immunology, Thomas Jefferson University, Philadelphia PA.
Chemokines are a major driver of lymphocyte migration via chemotaxis. Establishing chemokine profiles unique to specific sites and afflictions is vital to the investigation of lymphocyte trafficking. Murine Cytomegalovirus (MCMV) is a Beta-herpesvirus that induces a chronic infection utilizing the mouse submandibular glands (SMG) as a site of viral replication and shedding. It is hypothesized that multiple chemokines work in a redundant fashion to drive the migration of CD8+ T cells to the SMG of mice infected with MCMV. The chemotactic potential of chemokines was evaluated via ex vivo migration assays and phenotypes of CD8+ T cells were established via flow cytometry. Chemokines were selected by constitutive expression, known ability to induce chemotaxis in T cells and up-regulation in MCMV infected SMG’s via RNA seq analysis. While many chemokines were able to recruit CD8+ T cells, CD44hi (activated) CD8+ T cells and MCMV-specific CD8+ T cells at a statistically significant level, CXCL9, CXCL10, CXCL12 and CX3CL1 robustly recruited MCMV-specific CD8+ T cells. Expression of chemokine receptors on MCMV-specific CD8+ T cells in the SMG of infected mice relative to the spleen was evaluated by qPCR. Increased expression of CXCR3 and CXCR4, the receptors for CXCL9, CXCL10 and CXCL12 was seen on MCMV-specific CD8+ T cells from the SMG but negligible expression of CX3CL1’s receptor CX3CR1 was seen. These data suggest that CXCR3 and CXCR4 play a vital role in the migration of MCMV specific CD8+ T cells to infected SMG’s.
PEPTIDE RECOGNITION BY CD4+ T CELLS FROM MICE IMMUNIZED WITH VARIOUS RABIES VIRUS STRAINS.
GA Joseph, DC Hooper, Department of Cancer Biology, Thomas Jefferson University, Philadelphia PA, 19107, USA.
Rabies is an important public health issue with over 60,000 deaths and 15 million cases of post-exposure prophylaxis annually. The ability of Rabies Virus (RABV) to escape immune surveillance by expressing low levels of G protein and maintaining BBB integrity during infection exacerbates the effectiveness of currently available inactivated RABV vaccines, which induce a CD4+ T helper cell 2 (Th2) response and humoral immunity. Attenuated RABV-strains-induce CD4+ T helper cell 1 (Th1) cells which drive cell-mediated immunity and are vital in the clearance of RABV from CNS tissues. Based on the safety and efficacy of attenuated SPBNGAS RABV vaccine strains, we inserted the West Nile Virus (WNV) E gene into SPBNGAS-GAS to construct the SPBNGAS-WNV-GAS variant. The objective of this project was to determine if SPBNGAS variants and GAS-WNV-GAS induce comparable RABV-specific Th1-mediated immune responses and only the latter induces a response to WNV. We assessed the antigen specificity of CD4+ T cells from mice immunized with various strains of RABV. Immature DCs from naïve C57/BL6 mice were pulsed with immunodominant peptides derived from various RABV and WNV E antigenic sites and added to antigen specific-CD4+ T cells from mice immunized with SPBNGAS variants or GAS-WNV-GAS. The production of key cytokines by the T cells was then assessed. Results indicate that antigen specific-CD4+ T cells from mice immunized with SPBNGAS variants and GAS-WNV-GAS recognize RABV peptides consistently. In addition, CD4+ T cells from mice immunized with GAS-WNV-GAS identify both WNV E and RABV peptides. Furthermore, the results of serotyping for antigen-specific IgG1 and IgG2a confirms that infection with GAS-WNV-GAS elicits a Th1-dependent IgG2a response and therefore the same Th1 subset of CD4+ T cells as attenuated RABV infection.
UNMUTATED IGM ANTIBODIES to VI POLYSACCHARIDE OF SALMONELLA TYPHI CAN CONFER PROTECTIVE IMMUNITY IN A MURINE MODEL OF TYPHOID.
K. Pandya, K. Alugupalli
Dept. of Microbiology and Immunology, Thomas Jefferson University, Philadelphia, PA
T cell-dependent B cell responses develop in the germinal center, a specialized microenvironment in the secondary lymphoid organs. Antibodies generated during such responses undergo somatic hypermutation (SMH) and class-switch recombination (CSR) so therefore those antibodies are predominantly high-affinity IgG. In contrast, T cell-independent B cell responses do not form germinal centers; hence produce mainly IgM with no or very few mutations. To test the contribution of unmutated IgM antibodies in protective immunity, we immunized mice sufficient and deficient in activation-induced cytidine deaminase (AID), an enzyme required for both SHM and CSR, with heat-killed Salmonella Typhi, which expresses Vi Polysaccharide (ViPS). An anti-polysaccharide antibody response in the context of a bacterial immunization is considered a T cell-dependent process. Nevertheless, we found that wildtype and AID-/- mice immunized with heat killed S. Typhi produced comparable anti-ViPS IgM. To test the protective immunity coffered by this immunization, naïve and immunized wildtype and AID-/- mice were challenged with a chimeric S. Typhimurium expressing ViPS. Compared to naïve mice, immunized wildtype and AID-/- mice showed a significantly reduced bacterial burden in the blood, liver, and spleen. To test the role for unmutated IgM in the context of a bona fide T cell-independent B cell response, we have also evaluated the antibody response to a purified ViPS immunization. We found that both wildtype and AID-/- mice generated a significant anti-ViPS IgM response, which also correlated with a reduction in bacterial burden in the spleens of the immunized mice. This data indicates that unmutated IgM generated against ViPS can confer protective immunity.
DYSREGULATION OF NUCLEAR RECEPTOR ACTIVITY BY BISPHENOL-A (BPA) IN A DROSOPHILA MODEL.
M Vierick1, ST Smith2
1 Cell and Developmental Biology Program, Thomas Jefferson University, Philadelphia PA.
2 Department of Biology, Arcadia University, Glenside PA.
Bisphenol-A (BPA) is a high-production compound that has been reported to act as an endocrine disrupting chemical in several model systems. BPA elicits a non-monotonic dose response (NMDR) characteristic of several endocrine disrupting chemicals. The mechanisms underlying the NMDR are poorly understood but may be explained in part by the simultaneous dysregulation of multiple endocrine pathways. Drosophila was used as a model system to determine whether two endocrine pathways, the Estrogen Receptor Related (ERR) signaling pathway and the Ecdysone signaling pathway, could be simultaneously dysregulated by BPA. To this end, fat bodies from age-synchronized 3rd instar larvae that were treated with 10 uM BPA or control solutions were tested for expression of ERR target genes of the glycolytic pathway (pfk, hex-c, and gapdh). Although no expression differences were observed at 96 hours, the expression of these glycolytic enzymes was upregulated at 120h in the BPA treated larvae relative to control larvae. The activity of an ERR-responsive GFP reporter was also tested in salivary glands in BPA-treated (or non-treated) control larvae. BPA activated expression of the ERR-responsive GFP reporter at 120 but not 96 hours after egg laying (AEL), consistent with the endogenous gene expression of ERR target genes. Activation of the EcR pathway was also similarly assessed. Endogenous EcR target gene expression, including Broad, was found to be upregulated at 120 hours AEL, and BPA was found to weakly activate a (EcR)/Usp GFP-reporter line. These findings suggest that BPA exposure can simultaneously activate the ERR and EcR pathways, and the observed endpoints should be useful for assessing potential activation differences at lower and higher doses of BPA.
THERAPEUTIC TARGETING OF DSG2 IN SQUAMOUS CELL CARCINOMAS.
J Pierluissi1, M Mahoney1
1Department of Dermatology and Cutaneous Biology, Thomas Jefferson University, Philadelphia, PA
Monoclonal immunotherapy (mAb) is a powerful, widely utilized cancer therapeutic for multiple diseases. Successful treatments for malignant squamous cell carcinomas (SCCs) include the EGFR mAbs, cetuximab and panitumumab, as well as the EGFR-targeting small-molecule, erlotinib; however, these treatments often lead to treatment resistance and debilitating, acne-like skin rashes. Recent evidence suggests that consecutively treating or simultaneously targeting multiple signaling pathways can be more effective than a single treatment modality. We have shown that the desmosomal cadherin desmoglein 2 (Dsg2) is upregulated in SCCs and overexpression increases tumor development in transgenic mice. Furthermore, Dsg2 enhances EGFR level and activation both in vitro and in vivo. Loss of Dsg2 can inhibit tumorigenesis in vivo, thus we hypothesize that Dsg2 may be a potential target for mAb immunotherapy. Here we established tumor xenografts by injecting SCC cells (1x106) subcutaneously in severe combined immunodeficiency (SCID) mice to demonstrate that Dsg2 overexpression enhances tumor size and growth. Dsg2-directed mAb 6D8, which recognizes the extracellular domain 4 of Dsg2, down-regulated both Dsg2 and EGFR in cultured keratinocytes. Translating these findings to an in vivo model, intraperitoneal injection of affinity-purified 6D8 (5 mg/kg) twice weekly for 4 weeks reduced SCC xenograft size by 2-fold while eliciting no off-target side effects. Additionally, both Dsg2 and EGFR protein content were decreased in tumors of mice treated with Dsg2 mAb 6D8. These data illustrate that Dsg2-directed mAbs may be novel modalities that could be combined with anti-EGFR therapeutics to enhance the treatment of SCCs.
EFFECTS OF MEMBRANE PROTEIN PALMITOYLATION ON EXOSOME BIOGENESIS
Jessica M. Raad, Andrew M. Overmiller, James K. Wahl III, Mỹ G. Mahoney
Department of Dermatology and Cutaneous Biology, Thomas Jefferson University, Philadelphia PA.
Exosomes are membrane-derived extracellular vesicles that carry lipids, proteins, DNAs and RNAs that facilitate cell-cell communication, and have been implicated in many biological processes including wound healing and cancer. Protein palmitoylation is a reversible lipid modification that is important for protein trafficking and membrane association. Palmitoylation has been implicated in the formation of tetraspanin microdomain assembly that is critical for exosome formation. Here, we examined the role of palmitoylation of Dsg2, a transmembrane cadherin upregulated in epithelial-derived malignancies, in exosome biogenesis. We generated SCC cell lines stably expressing, by retroviral infection, GFP-labeled wildtype (WT) and palmitoylation-mutant Dsg2 proteins. Immunofluorescence illustrated that palmitoylation was critical for cell-cell membrane localization of Dsg2 and Cav1, further supporting the ability of Dsg2 to influence lipid raft dynamics. Cells were cultured in serum free media, and supernatant was collected for exosome purification by ultracentrifugation or polymer precipitation. Exosome concentration was determined by nanoparticle tracking analysis showing that while WT Dsg2 increased exosome release, mutations to any of the cysteine residues reduced exosome number with no correlation between the state of Dsg2 palmitoylation and exosome biogenesis. These results were confirmed by inhibiting palmitoylation with 2-bromopalmitate (2-BP), which decreased exosome release in Dsg2 overexpressing, but not parental cells. Future experiments are designed to assess the role of Dsg2 palmitoylation in association with tetraspanins and formation of tetraspanin complexes. The results will provide a mechanism by which Dsg2 modulates exosome biogenesis and 2-BP as a therapeutic strategy for Dsg2-overexpressing cancers.
OMEGA-3 POLYUNSATURATED FATTY ACIDS ATTENUATE ADIPOSE TISSUE INFLAMMATION: INHIBITION OF THE NLRP3 SIGNALING SYSTEM
K Roberts1, R Shah2
1Department of Cell and Developmental Biology, Thomas Jefferson University, Philadelphia PA.
2Department of Endocrinology, The Children’s Hospital of Philadelphia, Philadelphia PA.
Obesity and its related metabolic complications represent a state of low-grade inflammation, caused in part by recruitment of monocytes and macrophages into adipose tissue. The NLRP3 inflammasome, a regulator of innate immunity, has also been implicated in obesity-related inflammation. The fish oil derived long chain omega-3 polyunsaturated fatty acids (LC n-3 PUFA) eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have been shown to reduce obesity related inflammation, though the exact mechanisms have yet to be elucidated. The aim of this project is to confirm that EPA and DHA decrease adipose inflammatory response and identify the NLRP3 inflammasome as a potential target pathway through altered gene expression. Primary human adipocytes and THP1-derived macrophage were co-cultured and treated with EPA and/or DHA at a 100uM. Ex vivo treatment of subcutaneous and visceral adipose tissue from obese human subjects with EPA and DHA at 100uM and 200uM was also performed. qPCR was used to detect the expression of genes associated with the NLRP3 inflammasome (IL18, CASP1, IL7R, IL1b). Gene expression of IL18, CASP1, IL7R and IL1b was significantly decreased in both primary adipocytes and THP1 macrophages when treated with EPA and DHA. Expression of these genes were similarly decreased in both subcutaneous and visceral human adipose tissue ex vivo. These findings suggest that LC n-3 PUFAs may decrease adipose tissue inflammation by inhibiting the expression of the genes involved with NLRP3 inflammasome signaling system. There was no significant difference in expression when treated with EPA, DHA, or a combination of both, suggesting that the NLRP3 inflammasome may function as a common pathway for the mechanism of both.
RNA BINDING PROTEINS AND THEIR ROLE IN CANCER: CREATION OF A DATABASE
F. Ali and J. Brody, Thomas Jefferson University, Philadelphia, PA
The goal of this project is to create a database for scientists to use as a tool to and to explore the role of ribonucleic acid binding proteins (RNA-BPs) in all types cancer. Data from PubMed, Ensembl, Uniprot, and Toronto’s RNA-BP database were used to curate a searchable database in way that researches can identify trends and patterns based on their search criteria. The program Python was used to parse through data and return results. The result of the project is a Microsoft Excel table that can be used to identify which RNA-BPs are most often associated with (a) cancer in general (b) specific types of cancer. Furthermore, the Excel document provides basic information about each RNA-binding domain with 10 or more cancer articles associated with it such as RNA-binding domains, biological processes it is involved in, molecular functions, cellular components, pathways, and role in cancer. Of the original 492 RNA-binding proteins provided by Toronto’s RNA-BP Database, n were found to be significantly associated with cancer. The top ten are EWSR1, EPHA2, ELAVL1, IGF2BP3, FUS, LIN28A, SRSF1, TP63, and STIM1. Other findings illuminate that certain families of proteins, such as the Ephrin receptors, receptor tyrosine kinases, and xyz, tend to have RNA binding capabilities and are frequently found to play a role in tumorigenesis. The cancer types that have been most studied in relation to RNA-BPs are breast, colorectal, leukemia, liver, lung, ovarian, prostate, and sarcoma. All articles that discuss carcinogenesis/tumorigenesis in general or discuss multiple cancer types are categorized as “multiple/other”.
RETROSPECTIVE CASE-CONTROL STUDY OF TRAINING PROCESSES FOR ELECTRONIC DATA CAPTURE SYSTEMS
M Bocan1, J Pokorny2
1Department of Biomedical Sciences, Thomas Jefferson University, Philadelphia PA.
2Prevail Infoworks, Inc., Philadelphia PA
Electronic data capture (EDC) systems are a critical clinical trial tool to aggregate data and ensure data integrity. Comfortability and understanding of the EDC system is important in making sure that the results of a trial are clean and trustworthy. Currently, there is no work comparing the training techniques for EDC systems. It is hypothesized that there will be a shorter lag between training and system use for user’s who utilize an online training (OT) and they will produce less queries in the system than those who attended an investigator meeting (IM). The EDC of a global, multi-site clinical trial with two cohorts was used to collect the length of time between training and system use and the number of queries per subject for each site. Those trained via IM (n=83) acted as control and OT (n=149) as the case as IM is the standard practice. For both cohorts, the median time between training and login was significantly higher in IM than OT (C1: 71 v 7, C2: 50 v 6). However, the OT group had a significantly higher rate of query than the IM group (C1: 24 v 11, C2: 15 v 7). Additional analysis at the country level reiterates the same pattern with those with a longer time between training and login having a lower query rate. Preliminary ANOVA analysis shows that the number of queries per subject is potentially correlated with training type (p<0.01) and country (p<0.01) for the investigator. As anticipated, OT does occur closer to the start of system use than IM. However, in opposition to the hypothesis this is directly correlated with a higher number of queries indicating a lower rate of productivity for those who utilize online trainings over investigator meetings. Due to this correlation, online trainings should be re-evaluated and updated regularly to minimize any disparity in productivity.
THE EFFECTS OF DOXYCYCLINE ON METALLOPROTEINASE EXPRESSION AND SECRETED PROTEIN IN NORMAL AND AGED TENOCYTES.
RW Edwards1, R McBeath1,2
1 Orthopedic Research Division, Department of Orthopedic Surgery, Thomas Jefferson University, Philadelphia PA.
2 The Philadelphia Hand to Shoulder Center (formerly The Philadelphia Hand Center), Philadelphia PA
The purpose of this study is to determine if doxycycline and oxygen tension have differential effects on (1) MMP-1, MMP-2, MMP-9, TIMP-1, TIMP-2, collagen I and aggrecan gene expression (2) MMP-2 secretion and activity (3) Secretion of procollagen and fibronectin in tenocytes isolated from young and aged individuals. Doxycycline was administered to human tenocytes cultured in normoxia (21% O2) and hypoxia (1% O2) for four days. Media, cells and cellular mRNA were collected on day four and subjected to Live-Dead cell assay, qRT-PCR, gelatin zymography, MMP-2 activity assay and cell staining. In the present study, there was an observed difference in treatment response between young and aged cells in response to doxycycline and oxygen tension. Doxycycline was found to increase the gene expression and secretion of MMP-2, but had little effect on MMP-1 and MMP-9 gene expression independent of oxygen tension. The expression of TIMP-1 and TIMP-2 remained constant, however. Doxycycline decreased aggrecan gene expression in both young and aged tenocytes, in normoxia and hypoxia. Doxycycline increased collagen I gene expression in both cell types, but only young cells demonstrated increased secretion of procollagen, independent of oxygen tension. Fibronectin secretion was reduced in all doxycycline treated cells in normoxia and hypoxia. Interestingly, hypoxia induced MMP-1 and MMP-2 gene expression, increased MMP-2 secretion and activity, but decreased MMP-9 gene expression in all cell types. This study shows that the treatment response to doxycycline varies depending on the oxygen tension and cell type. Doxycycline alters the matrix production and degradative environment of the tenocyte, favoring collagen secretion in young tenocytes.
USE OF ETHIODOL AS A CARRIER OF TOPOTECAN FOR THE TREATMENT OF UVEAL MELANOMA.
L Butcher1, G Kaushal2
1Thomas Jefferson College of Biomedical Sciences, Philadelphia, PA.
2Department of Pharmaceutical Sciences, Thomas Jefferson College of Pharmacy, Philadelphia, PA
Approximately 50% of patients with uveal melanoma develop metastatic disease, with the liver as the most common site of metastatic tumors. Since many solid tumors express Topoisomerase I (Topo I), drugs that inhibit Topo I are rational choices for testing. Topotecan is a water-soluble analogue of camptothecin that inhibits Topo I. Ethiodol is an ethiodized oil known to concentrate in liver tumors and block liver tumor arteries, resulting in chemoembolization. Pairing of topotecan and Ethiodol could target the delivery of topotecan to the liver and minimize toxicity. However, an appropriate formulation of topotecan with Ethiodol is required. We formulated and investigated the release of topotecan from Ethiodol water-in-oil (w/o) and oil-in-water (o/w) emulsions with the intent of identifying a formulation that could deliver topotecan to liver tumor arteries. A liquid chromatography-mass spectrometry method was developed to quantitatively analyze topotecan. The partition coefficient of topotecan in Ethiodol was measured also. In vitro release studies of the emulsions were done using USP Dissolution Apparatus II. The o/w emulsions yielded immediate drug saturation in 6 hours. All w/o emulsions produced with hydrophilic-lipophilic balance 4.3-4.5 prolonged the drug release for 18 days. These results are a significant step towards overcoming the challenges associated with using an oil phase carrier (Ethiodol) for a water-soluble drug molecule (topotecan). An injectable drug delivery system creating a sustained drug release can offer an alternative treatment option that will provide a direct approach to treat uveal melanoma that has metastasized to the liver.
POLO-LIKE KINASE 3 ROLE IN WOUND-HEALING
RA Kilker1, U Naik 1*
1Cardeza Center for Hematology Research, Thomas Jefferson University, Philadelphia PA.
Non-healing wounds are a common cause of morbidity and mortality. Polo-like kinase 3 is a serine/threonine kinase that regulates cell-cycle activity through centrosome maturation, checkpoint recovery, spindle fiber assembly, cytokinesis and apoptosis. Previously publish research suggest that abnormalities in wound repair cells impairing cell-cycle activities, delays wound healing. This study aims to evaluate mice with the complete ablation of the Polo-like kinase 3 gene (PLK3), impact on wound healing. We investigate whether PLK3 KO mice suffer from a delayed contraction response. Mice wounds were generated using a 4mm biopsy punch. Mice were scarified on four designated time points to compare morphological differences across the stages of wound healing (day 2, day 4, day 8 and day 10). Tissue sections were analyzed through various histological and immunohistological techniques. Consisting of Hemotoxylin & Eosin, Masson’s Trichrome and Sirius Red. Tissue analysis will compare the rate of inflammation, collagen density, and differences in immature (collagen III) and mature (collagen I) at the wound site. We have found that our Polo-like kinase KO mice showed delayed wound closure with wounds healing ~12.4 days post injury ((N=5) P<0.05). Time points where significant difference in wound-healing are observed at Day 4, Day 9, and Day 10 post injury (Day 4, P<0.05; Day 9, P<0.05; Day 10, P<0.01).
Further Results Pending
Our findings indicate that the complete ablation of Polo-like kinase 3 leads to delayed wound closure.