Matching Items (130)
Description
Background: High risk types of human papillomavirus (HPV) are known to cause cancer, including cervical (99%) and oropharyngeal cancer (70%). HPV type 16 is the most common subtype. Three antigens that are critical for integration or tumor progression are E2, E6 and E7. In this study, we developed a systematic approach to identify naturally-processed HPV16-derived HLA class I epitopes for immunotherapy development. Methods: K562 cells, which lack HLA expression, were transduced with each HPV16 antigen using lentivirus and supertransfected with HLA-A2 by nucleofection. Stable cell lines expressing each antigen were selected for and maintained throughout the investigation. In order to establish a Gateway-compatible vector for robust transient gene expression, a Gateway recombination expression cloning cassette was inserted into the commercial Lonza pMAX GFP backbone, which has been experimentally shown to display high transfection expression efficiency. GFP was cloned into the vector and plain K562 cells were transfected with the plasmid by nucleofection. Results: Expression of K562-A2 was tested at various time points by flow cytometry and A2 expression was confirmed. Protein expression was shown for the transduced K562 E7 by Western blot analysis. High transfection efficiency of the pMAX_GFP_Dest vector (up to 97% GFP+ cells) was obtained 48 hours post transfection, comparable to the commercial GFP-plasmid. Conclusion: We have established a rapid system for target viral antigen co-expression with single HLA molecules for analysis of antigen presentation. Using HPV as a model system, our goal is to identify specific antigenic peptide sequences to develop immunotherapeutic treatments for HPV-associated cancers.
ContributorsVarda, Bianca Marie (Author) / Anderson, Karen (Thesis director) / Borges, Chad (Committee member) / Krishna, Sri (Committee member) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2016-05
Description
Identifying immunoreactive cytotoxic T lymphocytes (CTLs) by current technologies (cytokine secretion, intracellular cytokine, ELISPOT, and MHC tetramer assays) is often difficult when probing for multiple target antigens. CTLs activate and induce apoptosis of pathogenic cells when T-cell receptors (TCRs) specifically bind to antigenic peptides and major histocompatibility complexes (pMHCs) presented on the target cell’s surface. Flow cytometric MHC class I tetramer assays allow for the direct quantification and sorting of most CD8+ T lymphocytes whose TCRs recognize bound peptides, regardless of effector function. Class I tetramers are traditionally produced using BL21-DE3 E. coli expression, denaturation and folding in vitro, which is technically challenging, time-consuming, and low-throughput. We are developing an assay amenable to rapid, high-throughput screening of peptide libraries to characterize and quantitate antigen-specific CTLs in peripheral blood mononuclear cells (PBMCs). Baculovirus expression systems, utilizing host eukaryotic chaperones and isomerases, are capable of producing soluble, properly-folded protein complexes with high yields. The HLA-A*0201 heavy chain and beta-2-microglobulin genes were cloned into pIEx baculovirus expression vectors. Recombinant HLA-A*0201 and β2m viruses were synthesized using the BacMagic-3 DNA/pIEx method and transfected into Spodoptera frugiperda (Sf9) cells, and protein expression was confirmed by Western blot. To prepare T cells for testing, PBMCs from a healthy HLA-A2+ donor were collected and pulsed with DMSO control or CEF peptide pool (a mixture of CMV-, EBV-, and Flu-specific HLA class I epitopes). After 5 days, the CD8+ and CD8- fractions were sorted by MACS-based magnetic separation, and the frequency of FluM1-specific lymphocytes in the CD8+ populations was determined (0.1% of DMSO control vs. 0.772% of CEF-pulsed cells) using a commercial tetramer. We are optimizing HLA-A*0201 and β2m baculovirus co-infection ratios and evaluating the efficiency of intracellular MHC folding.
ContributorsRoesler, Alexander Scott (Author) / Anderson, Karen (Thesis director) / Blattman, Joseph (Committee member) / School of Molecular Sciences (Contributor) / School of Mathematical and Statistical Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2016-05
Description
Current studies in Multiple Myeloma suggest that patient tumors and cell lines cluster separately based on gene expression profiles. Hyperdiploid patients are also extremely underrepresented in established human myeloma cell lines (HMCLs). This suggests that the average HMCL model system does not accurately represent the average myeloma patient. To investigate this question we performed a combined CNA and SNV evolutionary comparison between four myeloma tumors and their established HMCLs (JMW-1, VP-6, KAS-6/1-KAS-6/2 and KP-6). We identified copy number changes shared between the tumors and their cell lines (mean of 74 events - 59%), those unique to patients (mean of 21.25 events - 17%), and those only in the cell lines (mean of 30.75 events \u2014 24%). A relapse sample from the JMW-1 patient showed 58% similarity to the primary diagnostic tumor. These data suggest that, on the level of copy number abnormalities, HMCLs show equal levels of evolutionary divergence as that observed within patients. By exome sequencing, patient tumors were 71% similar to their representative HMCLs, with ~12.5% and ~16.5% of SNVs unique to the tumors and HMCLs respectively. The HMCLs studied appear highly representative of the patient from which they were derived, with most differences associated with an enrichment of sub-populations present in the primary tumor. Additionally, our analysis of the KP-6 aCGH data showed that the patient's hyperdiploid karyotype was maintained in its respective HMCL. This discovery confirms the establishment and validation of a novel and potentially clinically relevant hyperdiploid HMCL that could provide a major advance in our ability to understand the pathogenesis and progression of this prominent patient population.
ContributorsBenard, Brooks Avery (Author) / Keats, Jonathan (Thesis director) / Anderson, Karen (Committee member) / Jelinek, Diane (Committee member) / School of Molecular Sciences (Contributor) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2016-05
Description
Colorectal cancer (CRC) is one of the most highly diagnosed cancers in the United States and accounts for 9.5% of all new cancer cases worldwide. With a 50% five-year prognosis, it is the second highest cancerous cause of death in the U.S. CRC tumors express antigens that are capable of inducing an immune response. The identification of autoantibodies (AAb) against tumor-associated antigens (TAA) may facilitate personalized tumor treatment in the form of targeted immunotherapy. The objective of this study was to observe the AAb expression raised against a 2000 human gene survey in late-stage colorectal cancer using the Nucleic Acid Programmable Protein Arrays (NAPPA). AAbs from serum samples were collected from 80 patients who died within 24 months of their last blood draw and 80 age and gender matched healthy control were profiled using NAPPA. TAA p53, a well-established protein that is one of the most highly mutated across a variety of cancers, was one of the top candidates based on statistical analysis, which, along with its family proteins p63 and p73 (which showed inverse AAb response profiles) warranted further testing via RAPID ELISA. Statistical analysis from these results revealed an inverse differential relationship between p53 and p63, in which p53 seropositivity was higher in patients than in controls, while the opposite was unexpectedly the case for p63. This study involving the AAb immunoprofiling of advanced stage CRC patients is one of the first to shed light on the high-throughput feasibility of immunoproteomic experiments using protein arrays as well as the identification of immunotherapy targets in a more rapid move towards specialized treatment of advanced CRC.
ContributorsSzeto, Emily (Author) / LaBaer, Joshua (Thesis director) / Qiu, Ji (Committee member) / Demirkan, Gokhan (Committee member) / Barrett, The Honors College (Contributor) / T. Denny Sanford School of Social and Family Dynamics (Contributor)
Created2014-12
Description
The focus of this project was to look at alternative treatments for endocrine resistant breast cancer (ERBC), which are breast cancers that have become resistant to hormone therapies such as Tamoxifen or aromatase inhibitors. The first part of this project involves investigating the relationship between histone de-acetylase inhibitor Vorinostat and Tamoxifen in MCF7 G11 cells, Tamoxifen resistant sub-clones, according to the PSOC Time grant. The second part involves targeting the androgen receptor (AR) in MCF7 sub-clones with AR antagonists, Bicalutamide and MDV3100, and investigating the possible usage of AR as a biomarker, due to over-expression of AR in ERBC, in accordance with the Mayo ASU Seed Grant.
The synergistic effects between Vorinostat and Tamoxifen observed through a phase II study on breast cancer patients resistant to hormone therapy may involve more than the modulation of ER-alpha to reverse Tamoxifen resistance in ERBC cells. RT-qPCR of genes expressed in Tamoxifen resistant cells, trefoil factor 1(TFF1) and v-myc avian myelocytomatosis viral oncogene homolog (MYC), were evaluated along with ESR1 and Diablo as a control. MYC was observed to have increased expression in the treated cells, whereas the other genes had a decrease in their expression levels after the cells were treated for 3 days with Vorinostat IC30 of 1 µM. As for targeting the AR, MCF7 Tamoxifen sensitive and resistant cells were not affected by the AR antagonists to determine an IC50. The cell viability for all MCF7 sub-clones only decreased for high concentrations of 5.56 µM - 50 µM in Bicalutamide and 16.67 µM – 50 µM of MDV1300. Furthermore, hormone depletion of MCF7 G11 Tamoxifen resistant sub-clones did not show a great response to DHT stimulation or the AR antagonists. In the RT-qPCR, the MCF7 G11 cells showed an increase in mRNA expression for ER, AR, and PR after 4 hours of treatment with estradiol. As for the DHT treatment, ER, AR, PR, and PSA had a minimal increase in the fold change, but the fold change in AR was less than in the estradiol treatment. The Mayo Clinic will investigate the possible usage of AR as a biomarker through immunohistochemistry.
The synergistic effects between Vorinostat and Tamoxifen observed through a phase II study on breast cancer patients resistant to hormone therapy may involve more than the modulation of ER-alpha to reverse Tamoxifen resistance in ERBC cells. RT-qPCR of genes expressed in Tamoxifen resistant cells, trefoil factor 1(TFF1) and v-myc avian myelocytomatosis viral oncogene homolog (MYC), were evaluated along with ESR1 and Diablo as a control. MYC was observed to have increased expression in the treated cells, whereas the other genes had a decrease in their expression levels after the cells were treated for 3 days with Vorinostat IC30 of 1 µM. As for targeting the AR, MCF7 Tamoxifen sensitive and resistant cells were not affected by the AR antagonists to determine an IC50. The cell viability for all MCF7 sub-clones only decreased for high concentrations of 5.56 µM - 50 µM in Bicalutamide and 16.67 µM – 50 µM of MDV1300. Furthermore, hormone depletion of MCF7 G11 Tamoxifen resistant sub-clones did not show a great response to DHT stimulation or the AR antagonists. In the RT-qPCR, the MCF7 G11 cells showed an increase in mRNA expression for ER, AR, and PR after 4 hours of treatment with estradiol. As for the DHT treatment, ER, AR, PR, and PSA had a minimal increase in the fold change, but the fold change in AR was less than in the estradiol treatment. The Mayo Clinic will investigate the possible usage of AR as a biomarker through immunohistochemistry.
ContributorsVorachitti, Merica (Author) / LaBaer, Joshua (Thesis director) / Anderson, Karen (Committee member) / Gonzalez, Laura (Committee member) / Barrett, The Honors College (Contributor) / School of Mathematical and Statistical Sciences (Contributor) / Department of Chemistry and Biochemistry (Contributor)
Created2014-05
Description
A coincidence reporter construct, consisting of the p21-promoter and two luciferase genes (Firefly and Renilla), was constructed for the screening of drugs that might inhibit Olig2's tumorigenic role in glioblastoma. The reporter construct was tested using an Olig2 inhibitor, HSP990, as well as short hairpin RNA targeting Olig2. Further confirmatory analysis is needed before the reporter cell line is ready for high-throughput screening at the NIH and lead compound selection.
ContributorsCusimano, Joseph Michael (Author) / LaBaer, Joshua (Thesis director) / Mangone, Marco (Committee member) / Mehta, Shwetal (Committee member) / Barrett, The Honors College (Contributor) / Department of Chemistry and Biochemistry (Contributor)
Created2014-05
Description
Background: The human papillomavirus (HPV) is the cause of virtually all cervical cancer, with over 520,000 new cases and 275,000 deaths annually. Although there are at least 200 unique HPV strains, only “high-risk” types, may progress to cancer. Serum antibodies to HPV oncoproteins are stable and specific markers that may be able to detect high-grade cervical intraepithelial neoplasia (CIN3). Biomarkers have potential as a rapid, point-of-care HPV screening tool for low resource areas in the way that traditional cytology cannot, and HPV DNA testing is not yet able to.
Methods: We have designed a multiplexed magnetics programmable bead ELISA (MagProBE) to profile the immune responses of the proteins from 11 high-risk HPV types and 2 low-risk types—106 genes in total. HPV genes were optimized for human expression and either built with PCR or commercially purchased, and cloned into the Gateway-compatible pANT7_cGST vector for in vitro transcription/translation (IVTT) in a MagProBE array. Anti-GST antibody (Ab) labeling was then used to measure gene expression.
Results: 53/106 (50%) HPV genes have been cloned and tested for expression of protein. 91% of HPV proteins expressed at levels above the background control (MFI = 2288), and the mean expression was MFI = 4318. Codon-optimized genes have also shown a 20% higher expression over non-codon optimized genes.
Conclusion: Although this research is ongoing, it suggests that gene optimization may improve IVTT expression of HPV proteins in human HeLa lysate. Once the remaining HPV proteins have been expression confirmed, the cDNA for each gene will be printed onto slides and tested in serologic assays to identify potential Ab biomarkers to CIN3.
Methods: We have designed a multiplexed magnetics programmable bead ELISA (MagProBE) to profile the immune responses of the proteins from 11 high-risk HPV types and 2 low-risk types—106 genes in total. HPV genes were optimized for human expression and either built with PCR or commercially purchased, and cloned into the Gateway-compatible pANT7_cGST vector for in vitro transcription/translation (IVTT) in a MagProBE array. Anti-GST antibody (Ab) labeling was then used to measure gene expression.
Results: 53/106 (50%) HPV genes have been cloned and tested for expression of protein. 91% of HPV proteins expressed at levels above the background control (MFI = 2288), and the mean expression was MFI = 4318. Codon-optimized genes have also shown a 20% higher expression over non-codon optimized genes.
Conclusion: Although this research is ongoing, it suggests that gene optimization may improve IVTT expression of HPV proteins in human HeLa lysate. Once the remaining HPV proteins have been expression confirmed, the cDNA for each gene will be printed onto slides and tested in serologic assays to identify potential Ab biomarkers to CIN3.
ContributorsResnik, Jack Isiah (Author) / Anderson, Karen (Thesis director) / Magee, Mitch (Committee member) / Purushothaman, Immanuel (Committee member) / Barrett, The Honors College (Contributor) / Department of Chemistry and Biochemistry (Contributor)
Created2013-05
Description
The long-term survival of patients with glioblastoma multiforme is compromised by the tumor's proclivity for local invasion into the surrounding normal brain. These invasive cells escape surgery and display resistance to chemotherapeutic- and radiation-induced apoptosis. We have previously shown that tumor necrosis factor-like weak inducer of apoptosis (TWEAK), a member of the tumor necrosis factor superfamily, can stimulate glioma cell invasion and survival via binding to the fibroblast growth factor-inducible 14 (Fn14) receptor and subsequent activation of the Rac1/NF-kappaB pathway. In addition, we have reported previously that Fn14 is expressed at high levels in migrating glioma cells in vitro and invading glioma cells in vivo. Here we demonstrate that TWEAK can act as a chemotactic factor for glioma cells, a potential process to drive cell invasion into the surrounding brain tissue. Specifically, we detected a chemotactic migration of glioma cells to the concentration gradient of TWEAK. Since Src family kinases (SFK) have been implicated in chemotaxis, we next determined whether TWEAK:Fn14 engagement activated these cytoplasmic tyrosine kinases. Our data shows that TWEAK stimulation of glioma cells results in a rapid phosphorylation of the SFK member Lyn as determined by multiplex Luminex assay and verified by immunoprecipitation. Immunodepletion of Lyn by siRNA oligonucleotides suppressed the chemoattractive effect of TWEAK on glioma cells. We hypothesize that TWEAK secretion by cells present in the glioma microenvironment induce invasion of glioma cells into the brain parenchyma. Understanding the function and signaling of the TWEAK-Fn14 ligand-receptor system may lead to development of novel therapies to therapeutically target invasive glioma cells.
ContributorsJameson, Nathan Meade (Author) / Anderson, Karen (Thesis director) / Lake, Douglas (Committee member) / Tran, Nhan (Committee member) / Barrett, The Honors College (Contributor) / School of Life Sciences (Contributor)
Created2013-05
Description
The pathogenesis of type 1 diabetes (T1D) is still not fully understood in the scientific community. Evidence has shown that viral infections are one of the important environmental factors associated with the disease development. Seven of the top T1D related viruses were selected to study the prevalence of viral humoral response in T1D patients using our innovative protein array platform called Nucleic Acid Programmable Protein Array (NAPPA). In this study, each viral gene was individually captured using various PCR based techniques, cloned into a protein expression vector, and assembled as the first version of T1D viral protein array. Humoral responses of IgG, IgA, and IgM were examined. Although each class of immunoglobulin generated a wide-range of reactivity, responses to various viral proteins from different proteins were observed. In summary, we captured most of the T1D related viral genes, established viral protein expression on the protein array, and displayed the serum response on the viral protein array. The successful progress will help to fulfill the long term goal of testing the viral infection hypothesis in T1D development.
ContributorsDavis, Amy Darlene (Author) / LaBaer, Joshua (Thesis director) / Qiu, Ji (Committee member) / Desi, Paul (Committee member) / Barrett, The Honors College (Contributor) / Department of Chemistry and Biochemistry (Contributor)
Created2013-05
Description
Our goal was to design a method to express soluble folded major histocompatibility complex (MHC) proteins using human cell line HeLa lysate with the novel 1-Step Human In Vitro Protein Expression by Thermo Scientific in the presence of β2 microglobulin (β2m) and antigenic peptide.
We confirmed that the soluble protein MHC-A2.1 could be successfully attached to the Luminex magnetic beads and detected using the primary antibody anti-GST and the detection antibody goat mAb mouse PE. The average net MFI of the attached pA2.1-bead complex was 8182. Biotinylated A2.1 MHC complexes pre-folded with β2m and FLU M1 peptide (A2.1 monomers) were also successfully attached to Luminex magnetic beads and detected with BB7.2. The average net MFI of the detected A2.1 monmer-bead complexes was 318. The protein MHC complexes were multimerized on magnetic beads to create MHC tetramers and detected with BB7.2, PE labeled monoclonal antibody, via median fluorescent intensity with the Luminex platform. Varying protein, β2 microglobulin (β2m), and peptide concentrations were tested in a number of MHC-A2.1 protein refolding trials. Different antigenic peptides and attachment methods were also tested. However, none of the MHC-A2.1 protein folding and capture trials were successful. Although MHC-A2.1 complexes and recombinant MHC molecules could be attached to Luminex magnetic beads and be detected by Luminex arrays, soluble protein A2.1 could not be successfully expressed, refolded, captured onto Luminex beads, and detected. All refolding trials resulted in a net MFI of <25. The failed refolding and capture trials of A2.1 lead to the conclusion that human cell line HeLa lysate cannot be used to properly fold MHC molecules. However, efforts to refold the complexes onto Luminex magnetic beads are ongoing. We are also using the baculovirus expression system to refold soluble A2.1 lysate onto peptide-bead complexes.
We confirmed that the soluble protein MHC-A2.1 could be successfully attached to the Luminex magnetic beads and detected using the primary antibody anti-GST and the detection antibody goat mAb mouse PE. The average net MFI of the attached pA2.1-bead complex was 8182. Biotinylated A2.1 MHC complexes pre-folded with β2m and FLU M1 peptide (A2.1 monomers) were also successfully attached to Luminex magnetic beads and detected with BB7.2. The average net MFI of the detected A2.1 monmer-bead complexes was 318. The protein MHC complexes were multimerized on magnetic beads to create MHC tetramers and detected with BB7.2, PE labeled monoclonal antibody, via median fluorescent intensity with the Luminex platform. Varying protein, β2 microglobulin (β2m), and peptide concentrations were tested in a number of MHC-A2.1 protein refolding trials. Different antigenic peptides and attachment methods were also tested. However, none of the MHC-A2.1 protein folding and capture trials were successful. Although MHC-A2.1 complexes and recombinant MHC molecules could be attached to Luminex magnetic beads and be detected by Luminex arrays, soluble protein A2.1 could not be successfully expressed, refolded, captured onto Luminex beads, and detected. All refolding trials resulted in a net MFI of <25. The failed refolding and capture trials of A2.1 lead to the conclusion that human cell line HeLa lysate cannot be used to properly fold MHC molecules. However, efforts to refold the complexes onto Luminex magnetic beads are ongoing. We are also using the baculovirus expression system to refold soluble A2.1 lysate onto peptide-bead complexes.
ContributorsChang, Peter S (Author) / Anderson, Karen (Thesis director) / Chang, Yung (Committee member) / Sundaresan, Krishna (Committee member) / Barrett, The Honors College (Contributor) / T. Denny Sanford School of Social and Family Dynamics (Contributor) / Department of Chemistry and Biochemistry (Contributor)
Created2013-05