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Upcoming MS Thesis Presentations with Abstracts
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.
MS Program Abstracts
DEVELOPMENT OF TOXICOLOGY TRAINING MODULES FOR EMERGENCY MEDICINE RESIDENTS
SM Muller, BS¹, KS Moran, MSc²
¹ New Jersey State Police Office of Forensic Science, Hammonton, NJ.
² Assistant Director, Forensic Science Program, Arcadia University, Glenside, PA.
Toxicology training modules were designed for physicians in the Thomas Jefferson University Hospital Emergency Medicine Residency program, with topic selection driven by a needs assessment. Recent statistics suggest that the likelihood of treating patients under the influence of drugs and/or alcohol has increased. Providing residents with current, up-to-date study modules on illicit drugs of abuse would be a useful addition to the emergency medicine (EM) residency training. The purpose of this project was to provide training modules to EM resident physicians. A needs assessment completed by the residents showed learning gaps in knowledge and application of information about drugs of abuse. The needs assessment indicated an interest and self-assessed need for additional information about drugs of abuse. Training modules were developed on three topics: heroin, synthetic cannabinoids, and synthetic cathinones. Residents’ knowledge about the three specific drugs of abuse was assessed prior to being given access to the modules and then again following completion of the modules. Data from the assessments have shown the three toxicology modules presented have had a positive impact and provided EM residents with additional knowledge for making informed decisions in patient-care.
Submitted by: Stephanie Muller
PROTEIN A CHROMATOGRAPHY LABORATORY SCALE RESIN REUSE STUDY.
R Gretchen¹, C Beck²
¹ Pharmaceutical Development and Manufacturing Sciences, Janssen R&D, Malvern, PA.
² Department of Pharmacology, Thomas Jefferson University, Philadelphia, PA.
Affinity chromatography has been used for many years in the biopharmaceutical industry but overtime, the chromatography resin’s performance can change and lose its effectiveness. Therefore, resin reuse studies are an important and necessary part of process validation for a monoclonal antibody purification process. This study was conducted to evaluate the performance of the Protein A resin (affinity chromatography resin) after cleaning with sodium hydroxide for 30 minutes and storing in a Sodium Acetate/Benzyl Alcohol, pH 5.0 buffer over a number of cycles. The study was concluded after 150 cycles. This resin cycling study was performed using a qualified scale down model of the Monoclonal X Protein A chromatography process. The following types of cycles were performed during this study: standard purification cycles (to achieve desired exposure to process/cleaning conditions but are not evaluated for quality), product evaluation purification cycles (monitor product quality), dynamic binding capacity cycles (monitor effective capacity), and viral clearance cycles (to monitor capacity of viral clearance). The performance characteristics that had associated acceptance criteria were asymmetry, yield, % monomer, residual protein A, residual DNA, residual HCP, viral clearance for an enveloped single-stranded RNA virus and viral clearance for a small non-enveloped single-stranded DNA virus. At the termination of the study the cycled column satisfied the acceptance criteria designated in the protocol for product quality. This study validated the cycling of Protein A resin in the Monoclonal X process for 150 cycles or 112 hours of sodium hydroxide contact time.
Submitted by: Rachel Gretchen
ELEVATED HOMOCYSTEINE LEVELS IN APP TRANSGENIC MICE CAUSE NEUROVASCULAR UNCOUPLING AS A RESULT OF OXIDATIVE STRESS.
MS Pearson¹, A Webber¹
¹ Merck Research Laboratories, West Point, PA
CHARACTERIZATION OF THE IMMUNE RESPONSE TO THE RABIES VIRUS SPBN-GAK
A Ejaz¹, DC Hooper².
¹ MS in Microbiology Program, Jefferson College of Biomedical Sciences,
² Department of Cancer Biology, Thomas Jefferson University, Philadelphia, PA 19107, USA
Rabies is a lethal disease caused by the rabies virus (RABV), a neurotropic virus, usually transmitted to humans through a bite or scratch from an infected animal. Once the virus reaches the CNS, via retrograde axonal transport, it causes fatal encephalitis. Research into rabies pathogenesis has led to the development of laboratory engineered rabies variants that are attenuated by directed mutations in the virus glycoprotein. This study is aimed at better characterizing the immune response to SPBN-GAK, a laboratory engineered variant that contains two opposing mutations in its glycoprotein sequence, one attenuating and a second that promotes pathogenicity. While the growth and spread properties of SPBN-GAK have been well defined in vitro, little is known about the immune response to this virus in vivo. Pathogenic RABV escape immune clearance through the maintenance of blood-brain barrier (BBB) integrity which prevents immune effector infiltration into CNS tissues. As SPBN-GAK carries an attenuating mutation, we hypothesize that the reemergence of the pathogenic phenotype may be due to the capacity of the virus to either avoid triggering BBB permeability or replicate too quickly for the immune response to control. To test these premises, mice were infected intranasally with SPBN-GAK and evaluated for BBB permeability as well as peripheral and CNS antiviral immunity. The majority (76%) of the mice died within 18 days of infection despite developing a normal-appearing rabies-specific immune response in the periphery. A slight increase in BBB fluid-phase permeability generally became evident after infection but only the survivors developed significant levels of rabies immunity in CNS tissues. This suggests that unlike fully pathogenic viruses, SPBN-GAK is pathogenic not by evading BBB permeability but by rapidly spreading to vital neurons before it can be contained by immune mechanisms infiltrating the CNS.
Submitted by: Aysha Ejaz
TYPE 2-BIASED ANTI-TUMOR IMMUNITY PROMOTES GLIOMA GROWTH IN A MOUSE MODEL.
S Sauma¹, DC Hooper
¹ Department of Neurological Surgery, Thomas Jefferson University, Philadelphia, PA, 19107, USA.
Regardless of whether or not their blood brain barrier (BBB) is intact, antibody reactive to tumor-derived exosomes is seen in the sera of primary astrocytoma patients yet to undergo surgery. Immunity to exosomal antigens with a bias towards type 2 over type 1 response patterns is associated with the accumulation of M2 macrophages and a worse prognosis in these patients. Immunotherapy aimed at targeting astrocytomas, including grade IV glioblastoma multiforme (GBM), must therefore surmount products of this pre-existing type 2 bias in order to achieve type 1-dependent tumoricidal immunity. To examine this premise, a C57BL/6 mouse model using syngeneic GL261 glioma cells was used to study the role of immune bias in glioma immunity. The addition of an anti-sense molecule targeting insulin-like growth factor 1 receptor (IGF1R) to GL261 cells or GL261-derived exosomes was found to promote an immune response that prevents glioma growth in C57BL/6 mice. This protective effect is not seen in mice lacking the Tbet transcription factor required for type 1 immunity indicating that type 1 mechanisms underlying an apparent type 2 response are indispensable. Moreover, when administered to mice following the implantation of low numbers of GL261 cells in the CNS, the largely type 2 response to exosome and cell-based vaccines results in greater tumor incidence compared to controls. Mice administered vaccine also show CD204 mRNA accumulation in the CNS suggesting that infiltration of type 2-associated M2 monocytes is enhanced. The possibility that M2 monocytes may promote tumor growth was investigated by examining the effects of treatment that reduces circulating CD163+ M2 cell numbers. When administered 19 days after implantation of GL261 cells in the flank, only several days before a tumor normally becomes palpable, this intervention dramatically arrests tumor development.
Submitted by: Sami Sauma
A RECOMBINANT RABIES VIRUS EXPRESSING B CELL ACTIVATING FACTOR INCREASES B CELL IMMUNOGENICITY WITHOUT INDUCING AUTOIMMUNITY.
SA Meuwissen¹, JP McGettigan²
¹ Department of Microbiology and Immunology, Thomas Jefferson University, Philadelphia PA.
² Department of Microbiology and Immunology, Thomas Jefferson University, Philadelphia PA.
Rabies is invariably fatal once symptoms develop and kills nearly 60,000 people per year. Current post-exposure prophylaxis (PEP) is complicated and costly, highlighting the need to develop a single-dose rabies vaccine. B cell Activating Factor (BAFF) is a potent cytokine that supports B cell functions. We exploited the functions of BAFF by constructing a recombinant RABV-based vaccine expressing BAFF (rRABV-BAFF). Mice immunized with rRABV-BAFF induced a more rapid and potent B cell response compared to mice immunized with rRABV alone. Despite these promising results, chronic BAFF expression has been linked to autoimmunity, including the development of autoantibodies, abnormal B cell compartments and delayed-type hypersensitivity (DTH) inflammatory responses. To rule out the potential for autoimmunity by rRABV-BAFF vaccination, mice were immunized with rRABV-BAFF or rRABV. Blood was collected as a source of serum and analyzed for the presence of anti-doublestranded DNA antibodies. Spleen and lymph node were collected as a source of B cells and analyzed for abnormal B cell compartments by flow cytometry analysis. No differences in the presence of autoantibodies or B cell compartments were noted in mice immunized with rRABV-BAFF compared with mice immunized with rRABV alone. Furthermore, no DTH reactions were noted in mice immunized with rRABV-BAFF. Together these data show that transient expression of BAFF from a live, attenuated rabies virus is able to promote an accelerated and robust B cell immune response without inducing autoimmunity, suggesting this approach may be exploited for further research in rabies vaccines.
Submitted by: Samantha Meuwissen
DEVELOPMENT OF DNA VACCINATION FOR IMMUN0-TARGETING OF UVEAL MELANOMA.
I Shnewer¹, A Vitali².
¹,²Department of Dermatology and Cutaneous Biology, Thomas Jefferson University, Philadelphia, PA.
Uveal melanoma (UM) is the deadliest and most aggressive cancer of the eye in adults. More than 80% of UM are characterized by the activating mutation, a substitution of Glutamine with Leucine at position 209 (Q209L) in two highly homologous G proteins – GNAQ and GNA11. We hypothesized that immune responses could be activated specifically against these mutated proteins. The primary goals of the study were to assess immunogenicity of the mutant proteins and develop a vaccination approach to elicit UM-specific immune response. MHC stabilization assay and synthetic peptides containing wild type (Q209) and mutant (L209) amino acids showed that Q209L substitution stabilizes MHC-peptide complex that could be presented to the T cells. Further, we generated a number of mtGNAQ-encoding plasmids using standard molecular cloning techniques. These plasmids encode mtGNAQ alone or fused in frame with universal non-natural pan DR (PADRE) and VP22 epitopes for additional activation of T-helper and cytotoxic T cells. These plasmids were used for the intradermal DNA vaccination of naïve C57BL6 mice under optimized previously conditions. After completion of vaccination protocol, splenic cells were collected and examined for the presence of the mtGNAQ-specific activated T cells by analyzing IFNg secretion using ELISpot assay. Furthermore, challenge of vaccinated mice with mtGNAQ-expressing cells led to the immune-mediated rejection of the challenge and inhibition of tumor growth.
Collectively, our studies demonstrated that: (I) Q209L peptides could be presented in the context of MHC class I; (II) Q209L-specific immune response can be induced via active vaccination; (III) adding of VP22 and PADRE epitopes to the vaccine increase its immunogenicity, and (IV) optimized vaccination induces potent immune response sufficient for the targeting of the mutated cells in vivo.
Submitted by: Inas Shnewer
THE EFFICACY OF INTERVENTIONAL PERIODONTAL TREATMENT TO MAINTAIN HEALTHY ORAL MICROFLORA.
A Nair, J Abrams¹ and N Amann¹
Thomas Jefferson University, Philadelphia, PA
¹ Abrams Center for Cosmetic Dentistry, Malvern, PA
The goal of this study is to examine the efficacy of interventional periodontal treatments. This was determined by analyzing the probing depths of patients with periodontal disease who maintained regular appointments. Periodontal disease is a multi factorial infection of the periodontium of the oral cavity. In a predictable and sequential process, bacteria colonize on the surface of the teeth. Early colonizers while compatible with periodontal health eventually pave the way for late colonizers, such as P. gingivalis, T. denticola and T. forsythia, which have been implicated in the progression of periodontal disease. Increasing proliferation of such pathogenic bacteria and their ability to invade epithelial gingival tissue results in periodontal infections, which have local and systemic implications. The health of the periodontium is determined during clinical visits by examining probing depths around each tooth and radiographic interpretation to analyze bone levels. Comparing patients who maintained regular appointments against those who did not, reveal the aggressive progression of periodontal disease and the benefits of consistent visits for treatments. Patients diagnosed with periodontal disease were divided into three groups: ‘gingivitis with regular appointments’, ‘gingivitis and localized periodontitis with regular appointments’, and ‘missed appointments’. Over the course of 2-3 visits, the probing depths are measured for each of these groups and significant improvements in periodontal health was observed in patients who maintained regular visits while patients who missed appointments revealed further progression of periodontal disease.
Submitted by: Anirudh Nair
JNK INHIBITION AND B1 INTEGRIN ABLATION UPON HYPOFRACTIONATED RADIATION PROMOTES PROSTATE TUMOR GROWTH AND CORRELATES WITH AN INCREASE IN FOCAL ADHESION KINASE EXPRESSION
D Deming¹ , L.R. Languino¹
¹ Sidney Kimmel Cancer Center, Department of Cancer Biology, Thomas Jefferson University, Philadelphia PA.
Radiation therapy is a key and effective modality for cancer treatment; however, prostate cancer (PrCa) patients invariably develop radiation resistant tumors. In previous studies, our laboratory has demonstrated that fractionated doses of ionizing radiation (10gy, every other week; total 50 Gy) significantly delays PrCa progression in transgenic adenocarcinoma of mouse prostate (TRAMP) mice. Similarly, our laboratory reports that a hypofractionated radiation treatment schedule (10Gy/day for 5 consecutive days) blocks prostate tumor growth in mice carrying a conditional ablation of b1 integrins in the prostatic epithelium (b1pc-/-/TRAMP) and in wild type (b1wt/TRAMP) mice. Administering the ATP competitive JNK inhibitor, SP600125 (SP), prevents radiation-induced tumor regression and allows the progression of invasive adenocarcinoma. The goal of this thesis was to analyze the expression and activity of Focal Adhesion Kinase (FAK) in irradiated tumors upon JNK inhibition. Utilizing Immunohistochemistry (IHC), ten specimens in each PPCES vehicle (PP) and SP (b1pc-/-/TRAMP) and five specimens in each PP and SP (b1wt/TRAMP) categories were analyzed for FAK intensity and scored in arbitrary units from 1-3. This study demonstrates that upon b1 integrin ablation and JNK inhibition, FAK expression is increased in the prostatic tumor cells in comparison to non-malignant prostate cells. Furthermore, the results show enhanced nuclear localization of FAK in SP-treated b1pc-/-/TRAMP mice as compared to those of the PP-treated cohort. In conclusion, we demonstrate a correlation of FAK induction with tumor progression upon irradiation and JNK inhibition in b1 null prostatic epithelium.
Submitted by: David Deming
INVESTIGATION OF THE LONG TERM STABILITY OF A CHIMERIC ANTIBODY (chIgG) THAT BINDS THE TERMINAL TELOPEPTIDE OF THE α2-CHAIN OF HUMAN COLLAGEN I.
BC Snyder¹ , A Fertala¹
¹ Department of Orthopaedic Surgery, Thomas Jefferson University, Philadelphia PA.
A mouse/human chimeric IgG-type (chIgG) antibody was developed to prevent fibrosis by the blocking of collagen fibril formation. The chIgG targets the C-terminal telopeptide of the α2-chain of human procollagen I. Once bound to the telopeptide the antibody inhibits the self-assembly of collagen molecules and the subsequent formation of collagen fibrils. To assess the in vivo efficacy of the antibody to limit post-traumatic joint stiffness, a surgery-induced arthrofibrosis rabbit model was employed. The current investigation focused on determining the long-term stability of the chIgG antibody at physiological range of temperatures. The hypothesis tested is that as the core temperature of a rabbit is high (38.3-39.4°C) the structural integrity of the chIgG would change over a 4-week incubation period. If this is the case, then it would reduce the effectiveness of the antibody to prevent fibrosis. To test this hypothesis, the effects of the raised temperature over 4-weeks on the stability and function of the chIgG were examined. Specifically, biosensor binding assays and thermal shift assays were employed to measure collagen binding and structural characteristics of the chIgG at physiological temperature of the rabbit. Results of these assays showed neither structural alterations nor significant changes in the binding affinity of the chIgG. Consequently, this study provides strong evidence for the long-term stability of the chIgG, indicating that it is an appropriate biologic for local preventative treatment of fibrotic lesions.
Submitted by: Bradley Snyder