SMARTech Collection: Department of Biomedical Engineering Theses and Dissertations

Glycoprotein-mediated interactions of dendritic cells with surfaces of defined chemistries

Tue, 29 May 2007 22:58:59 GMT

Title: Glycoprotein-mediated interactions of dendritic cells with surfaces of defined chemistries

Authors: Shankar, Sucharita P.

Abstract: Implanted combination devices comprising both biological as well as biomaterial components may trigger non-specific inflammatory responses against the biomaterial component as well as specific immune responses against the biological component. This specific immune response may be enhanced by the biomaterial, thereby implying a biomaterial-mediated adjuvant effect, or in contrast may be mitigated by the biomaterial. Since adjuvants function by triggering dendritic cell (DC) maturation, biomaterials may regulate DC responses and hence facilitate DC-orchestrated host responses. This research work has focused on examining DC responses to different model self-assembled monolayer (SAM) biomaterial chemistries, as an in vitro readout of the potential of these biomaterials to trigger DC maturation. The underlying hypothesis was that DCs recognize and respond to biomaterials either indirectly through the adsorbed protein layer, specifically through carbohydrate modifications of these proteins, or through carbohydrates inherent in the biomaterial chemistry, using PRRs to initiate an immune response. Towards this goal, DCs were derived from human peripheral blood mononuclear cells (PBMCs) by culture with DC differentiation cytokines and the culture systems were characterized as being composed of DCs as well as associated T and B lymphocytes. Culture of DCs on different SAM chemistries implied differential DC responses in terms of morphology, maturation marker expression and allostimulatory capacities as well as distinct underlying mechanisms responsible for these responses. Enzyme-linked lectin (ELLA) assays were used to characterize the profiles of carbohydrates associated with serum/plasma proteins adsorbed to different SAM chemistries. Differential profiles of DC carbohydrate ligands of CLRs were present on different chemistries. Furthermore, the profiles of human serum/plasma proteins adsorbed to and eluted from different SAM chemistries were assessed using immunoblot analysis. Finally, to observe the roles of carbohydrates in supporting DC maturation in the presence of a biomaterial, DCs were cultured in the presence of partially de-glycosylated FBS from which DC carbohydrate ligands were selectively removed. This research is significant towards the ultimate development of optimal design criteria for biomaterials for use in diverse tissue-engineering or vaccine development applications for which a wide spectrum of adjuvant effects are required.

Investigation of the limitations of viral gene transfer to murine embryonic stem cells

Sun, 18 May 2008 22:58:59 GMT

Title: Investigation of the limitations of viral gene transfer to murine embryonic stem cells

Authors: Chilton, Jamie Meredith

Abstract: Our objective was to address current cell source limitations in engineering pancreatic â-cells for the treatment of type 1 diabetes by investigating retroviral genetic modification of murine embryonic stem cells (mESC) with a murine stem cell virus (MSCV) encoding proendocrine transcription factor Neurogenin 3 (Ngn3). We found that expression of Ngn3 and the enhanced green fluorescent protein (eGFP) reporter gene were both significantly silenced in genetically modified mESCs. To overcome this obstacle and enhance the efficiency of retroviral gene transfer to mESCs in general, we employed a virus-polymer complexation method to deliver more transgenes to mESCs. Despite increased transgene delivery and integration in mESCs, transgene expression did not increase. Results suggest mESCs may be restricted in several steps of retrovirus transduction. We then investigated which steps of the virus lifecycle restrict efficient transduction of mESCs by using a recombinant MMuLV-derived retrovirus and a recombinant HIV-1-derived lentivirus to compare three major steps in the transduction of mESCs and NIH 3T3 cells - virus binding, virus integration, and transgene expression. We found that retroviruses and lentiviruses similarly bind 3 or 4-fold less efficiently to R1 mES cells than to NIH 3T3 fibroblasts. We also detected 3-fold fewer integrated retrovirus transgenes and 11-fold lower expression levels in NIH 3T3 cells, suggesting the primary limitation to retrovirus transduction may be low levels of transgene expression. In contrast we detected 10-fold fewer integrated lentivirus transgenes and 8-fold lower expression levels, suggesting lentivirus transduction may be limited by inefficient intracellular post-binding steps of transduction. We then investigated whether depletion of linker histone 1 in mESCs would alleviate silencing of retrovirus transgenes and improve gene transfer by transducing histone H1c, H1d, H1e triple null mESCs with different recombinant vectors. We found this did not improve viral gene transfer. This research is significant for improving protocols for gene transfer to ES cells and facilitating the use of modified ES cells in regenerative medicine.

Analysis of her2 testing in breast cancer: disparities, cost-effectiveness, and patterns of care

Tue, 30 Jun 2009 22:58:59 GMT

Title: Analysis of her2 testing in breast cancer: disparities, cost-effectiveness, and patterns of care

Authors: Ashok, Mahima

Abstract: HER2 breast cancer is an aggressive disease that occurs in 20 - 30% of the breast cancer population. Treatment for HER2 breast cancer includes use of an anti-HER2 monoclonal antibody, trastuzumab. Testing for HER2 is of critical importance due to the adverse side effects and substantial costs associated with this anti-HER2 treatment. Currently, two kinds of tests, Fluorescence In Situ Hybridization (FISH) and Immunohistochemistry (IHC), are FDA approved for determination of HER2 status in breast cancers. Clinical and non clinical factors that affect the choice HER2 test and the use of anti-HER2 therapy in breast cancer were analyzed using a data set containing information from six outpatient oncology clinics in the United States. The analysis showed that geographic location, cancer stage, and diagnosis date (pre- or post-publication of testing guidelines) have significant effects on choice of test. With regard to trastuzumab prescription, geographic location and HER2 status have significant effects on the prescription of trastuzumab. In addition, there was a non-significant trend for certain Medicare patients not to receive trastuzumab therapy. These findings indicate that disparities are present in breast cancer care based on geography and cancer stage, and highlight the importance of testing guidelines. The cost effectiveness of FISH vs. IHC was determined, by considering the financial and health-related costs associated with testing and subsequent treatment as well as the accuracy of each test. The results show that FISH is the optimal choice for HER2 testing and is more cost-effective than IHC.

Diffusion tensor imaging at long diffusion time

Mon, 29 Jun 2009 22:58:59 GMT

Title: Diffusion tensor imaging at long diffusion time

Authors: Rane, Swati

Abstract: Diffusion Tensor Imaging (DTI) is a well-established magnetic resonance technique that can non-invasively interpret tissue geometry and track neural pathways by sampling the diffusion of water molecules in the brain tissue. However, it is currently limited to tracking large nerve fiber bundles and fails to faithfully resolve thinner fibers. Conventional DTI studies use a diffusion time, t[subscript diff] of 30 ms - 55 ms for diffusion measurements. This work proposes the use of DTI at long t[subscript diff] to enhance the sensitivity of the method towards regions of low diffusion anisotropy and improve tracking of smaller fibers. The Stimulated Echo Acquisition Mode (STEAM) sequence was modified to allow DTI measurements at long t[subscript diff] (approximately 200 ms), while avoiding T2 signal loss. For comparison, DTI data was acquired using STEAM at the shorter value of t[subscript diff] and with the standard Double Spin Echo sequence with matched signal-to-noise ratio. This approach was tested on phantoms and fixed monkey brains and then translated to in vivo studies in rhesus macaques. Qualitative and quantitative comparison of the techniques was based on fractional anisotropy, diffusivity, three-phase plots and directional entropy. Tensor-field maps and probabilistic connectivity fronts were evaluated for all three acquisitions. Comparison of the tracked nerve pathways showed that fibers obtained at long t[subscript diff] were much longer. Further, the optic tract was tracked in ex vivo fixed rhesus brains for cross validation. The optic tract, traced at long t[subscript diff], conformed to the well documented anatomical description, thus confirming the accuracy of tract tracing at long t[subscript diff]. The benefits of DTI at long t[subscript diff] indeed help to realize the potential of tensor based tractography towards studying neural development and diagnosing neuro-pathologies, albeit the improvement is more significant ex vivo than in vivo.