Matching Items (6)
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- All Subjects: Biomarkers
- Creators: Anderson, Karen
Description
Introduction: Human papillomavirus (HPV) infection is seen in up to 90% of cases of cervical cancer, the third leading cancer cause of death in women. Current HPV screening focuses on only two HPV types and covers roughly 75% of HPV-associated cervical cancers. A protein based assay to test for antibody biomarkers against 98 HPV antigens from both high and low risk types could provide an inexpensive and reliable method to screen for patients at risk of developing invasive cervical cancer. Methods: 98 codon optimized, commercially produced HPV genes were cloned into the pANT7_cGST vector, amplified in a bacterial host, and purified for mammalian expression using in vitro transcription/translation (IVTT) in a luminescence-based RAPID ELISA (RELISA) assay. Monoclonal antibodies were used to determine immune cross-reactivity between phylogenetically similar antigens. Lastly, several protein characteristics were examined to determine if they correlated with protein expression. Results: All genes were successfully moved into the destination vector and 86 of the 98 genes (88%) expressed protein at an adequate level. A difference was noted in expression by gene across HPV types but no correlation was found between protein size, pI, or aliphatic index and expression. Discussion: Further testing is needed to express the remaining 12 HPV genes. Once all genes have been successfully expressed and purified at high concentrations, DNA will be printed on microscope slides to create a protein microarray. This microarray will be used to screen HPV-positive patient sera for antibody biomarkers that may be indicative of cervical cancer and precancerous cervical neoplasias.
ContributorsMeshay, Ian Matthew (Author) / Anderson, Karen (Thesis director) / Magee, Mitch (Committee member) / Katchman, Benjamin (Committee member) / Barrett, The Honors College (Contributor) / School of Life Sciences (Contributor)
Created2015-05
Description
Background: Recent interests in continuous biomonitoring and the surge of wearable biotechnology demand a better understanding of sweat as a noninvasive biomarker resource. The ability to use sweat as a biofluid provides the opportunity for noninvasive early and continuous diagnostics. This thesis serves to help fill the existing knowledge gap in sweat biomarker discovery and applications.
Experimental Design: In part one of this study, exercise-induced eccrine sweat was collected from 50 healthy individuals and analyzed using mass spectrometry, protein microarrays, and quantitative ELISAs to identify a broad range of proteins, antibody isotypes, and cytokines in sweat. In part two of this study, cortisol and melatonin levels were analyzed in exercise-induced sweat and plasma samples collected from 22 individuals.
Results: 220 unique proteins were identified by shotgun analysis in pooled sweat samples. Detectable antibody isotypes include IgA (100% positive; median 1230 ± 28 700 pg/mL), IgD (18%; 22.0 ± 119 pg/mL), IgG1 (96%;1640 ± 6750 pg/mL), IgG2 (37%; 292 ± 6810 pg/mL), IgG3 (71%;74.0 ± 119 pg/mL), IgG4 (69%; 43.0 ± 42.0 pg/mL), and IgM (41%;69.0 ± 1630 pg/mL). Of 42 cytokines, three were readily detected in all sweat samples (p<0.01). The median concentration for interleukin-1α was 352 ± 521 pg/mL, epidermal growth factor was 86.5 ± 147 pg/mL, and angiogenin was 38.3 ± 96.3 pg/mL. Multiple other cytokines were detected at lower levels. The median and standard deviation of cortisol was determined to be 4.17 ± 11.1 ng/mL in sweat and 76.4 ± 28.8 ng/mL in plasma. The correlation between sweat and plasma cortisol levels had an R-squared value of 0.0802 (excluding the 2 highest sweat cortisol levels). The median and standard deviation of melatonin was determined to be 73.1 ± 198 pg/mL in sweat and 194 ± 93.4 pg/mL in plasma. Similar to cortisol, the correlation between sweat and plasma melatonin had an R-squared value of 0.117.
Conclusion: These studies suggest that sweat holds more proteomic and hormonal biomarkers than previously thought and may eventually serve as a noninvasive biomarker resource. These studies also highlight many of the challenges associated with monitoring sweat content including differences between collection devices and hydration, evaporation losses, and sweat rate.
Experimental Design: In part one of this study, exercise-induced eccrine sweat was collected from 50 healthy individuals and analyzed using mass spectrometry, protein microarrays, and quantitative ELISAs to identify a broad range of proteins, antibody isotypes, and cytokines in sweat. In part two of this study, cortisol and melatonin levels were analyzed in exercise-induced sweat and plasma samples collected from 22 individuals.
Results: 220 unique proteins were identified by shotgun analysis in pooled sweat samples. Detectable antibody isotypes include IgA (100% positive; median 1230 ± 28 700 pg/mL), IgD (18%; 22.0 ± 119 pg/mL), IgG1 (96%;1640 ± 6750 pg/mL), IgG2 (37%; 292 ± 6810 pg/mL), IgG3 (71%;74.0 ± 119 pg/mL), IgG4 (69%; 43.0 ± 42.0 pg/mL), and IgM (41%;69.0 ± 1630 pg/mL). Of 42 cytokines, three were readily detected in all sweat samples (p<0.01). The median concentration for interleukin-1α was 352 ± 521 pg/mL, epidermal growth factor was 86.5 ± 147 pg/mL, and angiogenin was 38.3 ± 96.3 pg/mL. Multiple other cytokines were detected at lower levels. The median and standard deviation of cortisol was determined to be 4.17 ± 11.1 ng/mL in sweat and 76.4 ± 28.8 ng/mL in plasma. The correlation between sweat and plasma cortisol levels had an R-squared value of 0.0802 (excluding the 2 highest sweat cortisol levels). The median and standard deviation of melatonin was determined to be 73.1 ± 198 pg/mL in sweat and 194 ± 93.4 pg/mL in plasma. Similar to cortisol, the correlation between sweat and plasma melatonin had an R-squared value of 0.117.
Conclusion: These studies suggest that sweat holds more proteomic and hormonal biomarkers than previously thought and may eventually serve as a noninvasive biomarker resource. These studies also highlight many of the challenges associated with monitoring sweat content including differences between collection devices and hydration, evaporation losses, and sweat rate.
ContributorsZhu, Meilin (Author) / Anderson, Karen (Thesis director) / Blain Christen, Jennifer (Committee member) / Gronowski, Ann (Committee member) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2019-05
Description
Human papillomavirus (HPV) is the causative agent of cervical cancer. Persistent infection with high-risk HPV 16, 18 or 45 species is associated with the development and progression of cervical cancer. HPV genotyping and Pap smear tests are the regular methods used to detect pre-invasive cervical lesions, but there is a need for developing a rapid biomarker to profile immunity to these viruses. The viral E7 oncogene is expressed in most HPV-associated cancers and anti-E7 antibodies can be detected in the blood of patients with cervical cancer. This research was focused on viral E7 oncogene expression to be used in development of low-cost point of care tests, enabling patients from low resource settings to detect the asymptotic stage of cervical cancer and be able to seek treatment early. In order to produce the E7 protein in vitro to measure antibody levels, GST tagged E7 genes from HPV 16, 18 and 45 species were inserted into the pDEST15 vector and expressed in E. coli BL21DE3 cells that were induced with 1mM of IPTG. The E7-GST fused expressed protein was then purified using glutathione beads and resolved on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Protein expression was 5.8 \u03bcg/ml for HPV 16E7 in 500 ml culture and for the 500 ml culture of HPV 18 E7 and 45 E7 were 10.5 \u03bcg/ml and 10.5 \u03bcg/ml for HPV 18E7 and 45E7 respectively. High yield values are showing high expression levels of GST-tagged E7 recombinant protein which can be used for serotyping a number of individuals. This shows that HPV E7 can be produced in large quantities that can potentially be used in point of care tests that can help identify women at risk of cervical cancer. In conclusion, the E7 protein produced in this study can potentially be used to induce humoral responses in patients\u2019 sera for understanding the immune response of cervical cancer.
ContributorsMakuyana, Ntombizodwa (Author) / Anderson, Karen (Thesis director) / Ewaisha, Radwa (Committee member) / Varsani, Arvind (Committee member) / Hou, Ching-Wen (Committee member) / School of Mathematical and Statistical Sciences (Contributor) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2018-12
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
Oropharyngeal cancer (OPC) is the world's sixth most common cancer and in many cases is associated with infection with human papillomavirus (HPV) type 16. Antibodies (Abs) to HPV16 viral antigens are potential diagnostic biomarkers of HPV-associated OPC (HPV OPC). A custom multiplexed bead array assay was used to detect Abs to HPV16 antigens E1, CE2, NE2, E4, E5, E6, E7, L1, and L2. Following extensive optimization of the assay, these genes were expressed as GST-fusion proteins and captured onto anti-GST magnetic beads. Serum was obtained from 256 OPC patients at the time of diagnosis and from 78 healthy controls. The median fluorescent intensity (MFI) was determined for each antigen and ratios of MFI to control GST-fusion protein were determined for each serum sample. Cutoff values were set as the mean + 3 SD of the MFIs of healthy controls and p-values were calculated using Wilcoxon unpaired and Fisher's exact test. Results of this experiment showed that HPV16 E1, CE2, NE2, E4, E6, and E7 Ab levels were elevated in OPC patients compared to controls (p<0.001), as were Ab levels to L1 (p = 0.013) and L2 (p = 0.023), per Fischer's exact test. Abs to CE2, NE2, E6, and E7 were identified as a potential biomarker panel for early detection of HPV OPC. For the 111 patients with known HPV+ tumors as measured by tumor PCR of E6 and/or E7, this assay had a sensitivity of 90% and specificity of 87% (AUC = 0.96). From these results, we conclude that custom bead array assays can be used to detect HPV16 Abs in patient sera, and we have identified a 4-Ab biomarker panel for the early detection of HPV OPC.
ContributorsGoulder, Alison Leigh (Author) / Anderson, Karen (Thesis director) / Lake, Douglas (Committee member) / Cheng, Julia (Committee member) / Barrett, The Honors College (Contributor) / School of Life Sciences (Contributor)
Created2013-05
Description
Glioblastoma (GBM) is a highly invasive and deadly late stage tumor that develops from abnormal astrocytes in the brain. With few improvements in treatment over many decades, median patient survival is only 15 months and the 5-year survival rate hovers at 6%. Numerous challenges are encountered in the development of treatments for GBM. The blood-brain barrier (BBB) serves as a primary obstacle due to its innate ability to prevent unwanted molecules, such as most chemotherapeutics, from entering the brain tissue and reaching malignant cells. The GBM cells themselves serve as a second obstacle, having a high level of genetic and phenotypic heterogeneity. This characteristic improves the probability of a population of cells to have resistance to treatment, which ensures the survival of the tumor. Here, the development and testing of two different modes of therapy for treating GBM is described. These therapeutics were enhanced by pathogenic peptides known to improve entry into brain tissue or to bind GBM cells to overcome the BBB and/or tumor cell heterogeneity. The first therapeutic utilizes a small peptide, RVG-29, derived from the rabies virus glycoprotein to improve brain-specific delivery of nanoparticles encapsulated with a small molecule payload. RVG-29-targeted nanoparticles were observed to reach the brain of healthy mice in higher concentrations 2 hours following intravenous injection compared to control particles. However, targeted camptothecin-loaded nanoparticles were not capable of producing significant treatment benefits compared to non-targeted particles in an orthotopic mouse model of GBM. Peptide degradation following injection was shown to be a likely cause for reduced treatment benefit. The second therapeutic utilizes chlorotoxin, a non-toxic 36-amino acid peptide found in the venom of the deathstalker scorpion, expressed as a fusion to antibody fragments to enhance T cell recognition and killing of GBM. This candidate biologic, known as anti-CD3/chlorotoxin (ACDClx) is expressed as an insoluble protein in Nicotiana benthamiana and Escherichia coli and must be purified in denaturing and reducing conditions prior to being refolded. ACDClx was shown to selectively activate T cells only in the presence of GBM cells, providing evidence that further preclinical development of ACDClx as a GBM immunotherapy is warranted.
ContributorsCook, Rebecca Leanne (Author) / Blattman, Joseph N (Thesis advisor) / Sirianni, Rachael W. (Thesis advisor) / Mor, Tsafrir (Committee member) / Anderson, Karen (Committee member) / Arizona State University (Publisher)
Created2019