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- All Subjects: Immunology
- Creators: School of Molecular Sciences
Description
Breast cancer is the leading cause of cancer-related deaths of women in the united states. Traditionally, Breast cancer is predominantly treated by a combination of surgery, chemotherapy, and radiation therapy. However, due to the significant negative side effects associated with these traditional treatments, there has been substantial efforts to develop alternative therapies to treat cancer. One such alternative therapy is a peptide-based therapeutic cancer vaccine. Therapeutic cancer vaccines enhance an individual's immune response to a specific tumor. They are capable of doing this through artificial activation of tumor specific CTLs (Cytotoxic T Lymphocytes). However, in order to artificially activate tumor specific CTLs, a patient must be treated with immunogenic epitopes derived from their specific cancer type. We have identified that the tumor associated antigen, TPD52, is an ideal target for a therapeutic cancer vaccine. This designation was due to the overexpression of TPD52 in a variety of different cancer types. In order to start the development of a therapeutic cancer vaccine for TPD52-related cancers, we have devised a two-step strategy. First, we plan to create a list of potential TPD52 epitopes by using epitope binding and processing prediction tools. Second, we plan to attempt to experimentally identify MHC class I TPD52 epitopes in vitro. We identified 942 potential 9 and 10 amino acid epitopes for the HLAs A1, A2, A3, A11, A24, B07, B27, B35, B44. These epitopes were predicted by using a combination of 3 binding prediction tools and 2 processing prediction tools. From these 942 potential epitopes, we selected the top 50 epitopes ranked by a combination of binding and processing scores. Due to the promiscuity of some predicted epitopes for multiple HLAs, we ordered 38 synthetic epitopes from the list of the top 50 epitope. We also performed a frequency analysis of the TPD52 protein sequence and identified 3 high volume regions of high epitope production. After the epitope predictions were completed, we proceeded to attempt to experimentally detected presented TPD52 epitopes. First, we successful transduced parental K562 cells with TPD52. After transduction, we started the optimization process for the immunoprecipitation protocol. The optimization of the immunoprecipitation protocol proved to be more difficult than originally believed and was the main reason that we were unable to progress past the transduction of the parental cells. However, we believe that we have identified the issues and will be able to complete the experiment in the coming months.
ContributorsWilson, Eric Andrew (Author) / Anderson, Karen (Thesis director) / Borges, Chad (Committee member) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2016-05
Description
Abstract:
Background: Chronic rhinosinusitis (CRS) is defined as symptomatic inflammation of the nose and paranasal sinuses lasting more than 12 weeks. Persistent inflammation is thought to originate from multiple factors including host physical and innate barrier defects and the exposure of the sinonasal mucosa to exogenous microorganisms. Regional differences in the innate host defense molecules present in nasal and sinus tissue have been recently reported. Thus, a histopathological study was conducted by Lal et al. to compare inflammatory changes in the ethmoid sinus mucosa and nasal turbinate tissue for CRS patients and controls. The objective of this work was to interpret the histopathological data from an immunobiological perspective and describe the significance of the results within the context of current scientific literature.
Methods: Tissue samples were collected from sinonasal surgery patients in three specific regions: ethmoid cells ± uncinate process (EC) in all patients and the inferior (IT) or middle turbinate (MT). EC and IT/MT samples were compared using Cohen’s kappa coefficient to measure agreement based on overall severity of inflammation, eosinophil count per high power field, and the predominant inflammatory cell infiltrate. The results of this study were compared with the current cohort of scientific literature regarding CRS pathogenesis. Both previous and current hypotheses were considered to construct a holistic overview of the development of the current understanding of CRS.
Results: The histopathology study determined that regional differences in degree and type of inflammation may be present in the nose and paranasal cavity. These findings support the current understanding of CRS as an inflammatory disease that is likely mediated by both host and environmental factors.
Conclusions: The histopathology study supports the current cohort of CRS research and provides evidence in support of the involvement of host factors in CRS pathogenesis.
Background: Chronic rhinosinusitis (CRS) is defined as symptomatic inflammation of the nose and paranasal sinuses lasting more than 12 weeks. Persistent inflammation is thought to originate from multiple factors including host physical and innate barrier defects and the exposure of the sinonasal mucosa to exogenous microorganisms. Regional differences in the innate host defense molecules present in nasal and sinus tissue have been recently reported. Thus, a histopathological study was conducted by Lal et al. to compare inflammatory changes in the ethmoid sinus mucosa and nasal turbinate tissue for CRS patients and controls. The objective of this work was to interpret the histopathological data from an immunobiological perspective and describe the significance of the results within the context of current scientific literature.
Methods: Tissue samples were collected from sinonasal surgery patients in three specific regions: ethmoid cells ± uncinate process (EC) in all patients and the inferior (IT) or middle turbinate (MT). EC and IT/MT samples were compared using Cohen’s kappa coefficient to measure agreement based on overall severity of inflammation, eosinophil count per high power field, and the predominant inflammatory cell infiltrate. The results of this study were compared with the current cohort of scientific literature regarding CRS pathogenesis. Both previous and current hypotheses were considered to construct a holistic overview of the development of the current understanding of CRS.
Results: The histopathology study determined that regional differences in degree and type of inflammation may be present in the nose and paranasal cavity. These findings support the current understanding of CRS as an inflammatory disease that is likely mediated by both host and environmental factors.
Conclusions: The histopathology study supports the current cohort of CRS research and provides evidence in support of the involvement of host factors in CRS pathogenesis.
ContributorsElwell, Zachary Andrew (Author) / Blattman, Joseph (Thesis director) / Bean, Heather (Committee member) / Lal, Devyani (Committee member) / School of International Letters and Cultures (Contributor) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2017-05
Description
Rasopathies are a family of developmental syndromes that exhibit craniofacial abnormalities, cognitive disabilities, developmental delay and increased risk of cancer. However, little is known about the pathogenesis of developmental defects in the nervous system. Frequently, gain-of-function mutations in the Ras/Raf/MEK/ERK cascade (aka ERK/MAPK) are associated with the observed pathogenesis. My research focuses on defining the relationship between increased ERK/MAPK signaling and its effects on the nervous system, specifically in the context of motor learning. Motor function depends on several neuroanatomically distinct regions, especially the spinal cord, cerebellum, striatum, and cerebral cortex. We tested whether hyperactivation of ERK/MAPK specifically in the cortex was sufficient to drive changes in motor function. We used a series of genetically modified mouse models and cre-lox technology to hyperactivate ERK/MAPK in the cerebral cortex. Nex:Cre/NeuroD6:Cre was employed to express a constitutively active MEK mutation throughout all layers of the cerebral cortex from an early stage of development. RBP4:Cre, caMEK only exhibited hyper activation in cortical glutamatergic neurons responsible for cortical output (neurons in layer V of the cerebral cortex). First, the two mouse strains were tested in an open field paradigm to assess global locomotor abilities and overall fitness for fine motor tasks. Next, a skilled motor reaching task was used to evaluate motor learning capabilities. The results show that Nex:Cre/NeuroD6:Cre, caMEK mutants do not learn the motor reaching task, although they performed normally on the open field task. Preliminary results suggest RBP4:Cre, caMEK mutants exhibit normal locomotor capabilities and a partial lack of learning. The difference in motor learning capabilities might be explained by the extent of altered connectivity in different regions of the corticospinal tract. Once we have identified the neuropathological effects of various layers in the cortex we will be able to determine whether therapeutic interventions are sufficient to reverse these learning defects.
ContributorsRoose, Cassandra Ann (Author) / Newbern, Jason M. (Thesis director) / Olive, Foster (Committee member) / Bjorklund, Reed (Committee member) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2016-12
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 RAS/MAPK (RAS/Mitogen Activated Protein Kinase) pathway is a highly conserved, canonical signaling cascade that is highly involved in cellular growth and proliferation as well as cell migration. As such, it plays an important role in development, specifically in development of the nervous system. Activation of ERK is indispensable for the differentiation of Embryonic Stem Cells (ESC) into neuronal precursors (Li z et al, 2006). ERK signaling has also shown to mediate Schwann cell myelination of the peripheral nervous system (PNS) as well as oligodendrocyte proliferation (Newbern et al, 2011). The class of developmental disorders that result in the dysregulation of RAS signaling are known as RASopathies. The molecular and cell-specific consequences of these various pathway mutations remain to be elucidated. While there is evidence for altered DNA transcription in RASopathies, there is little work examining the effects of the RASopathy-linked mutations on protein translation and post-translational modifications in vivo. RASopathies have phenotypic and molecular similarities to other disorders such as Fragile X Syndrome (FXS) and Tuberous Sclerosis (TSC) that show evidence of aberrant protein synthesis and affect related pathways. There are also well-defined downstream RAS pathway elements involved in translation. Additionally, aberrant corticospinal axon outgrowth has been observed in disease models of RASopathies (Xing et al, 2016). For these reasons, this present study examines a subset of proteins involved in translation and translational regulation in the context of RASopathy disease states. Results indicate that in both of the tested RASopathy model systems, there is altered mTOR expression. Additionally the loss of function model showed a decrease in rps6 activation. This data supports a role for the selective dysregulation of translational control elements in RASopathy models. This data also indicates that the primary candidate mechanism for control of altered translation in these modes is through the altered expression of mTOR.
ContributorsHilbert, Alexander Robert (Author) / Newbern, Jason (Thesis director) / Olive, M. Foster (Committee member) / Bjorklund, Reed (Committee member) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2017-05
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
P2RX7 promotes a pro-memory signature in effector CD8+ T cells dependent on Zeb2 negative regulation
Description
Memory CD8+ T cells protect against secondary viral infections. They develop and maintain exclusively in circulation (e.g. central memory - Tcm) or are excluded from re-circulation (resident memory - Trm). The extracellular ATP receptor P2RX7 promotes both Tcm and Trm generation. High (P2RX7hi) P2RX7-expressing early effector cells show survival, memory and pluripotency genes. Conversely, many terminal effector (TE) and apoptosis genes are upregulated in low (P2RX7lo) P2RX7-expressing cells. Among these genes is the zinc-finger transcriptional repressor Zeb2, which promotes TE differentiation at the expense of the memory cell pool. Given that Zeb2 was higher in P2RX7lo early effector cells, we postulated that Zeb2 ablation would allow P2RX7-deficient CD8+ T cells to skew towards memory subsets. To test this, we used RNP-based CRISPR-Cas9 to knockout Zeb2 in wild type or P2RX7-deficient P14 cells. At the memory timepoint, Zeb2 ablation led to a rescue of the ability of P2RX7-deficient cells to differentiate into the CD62L+ Tcm and CD69hiCD103hi Trm subsets, as well as increase the population of each. Our data suggest that P2RX7 imprints a pro-memory signature that is, to some extent, dependent on the negative regulation of Zeb2.
ContributorsVan Dijk, Sarah (Author) / Holechek, Susan (Thesis director) / Borges da Silvs, Henrique (Committee member) / Barrett, The Honors College (Contributor) / School of Life Sciences (Contributor) / School of Molecular Sciences (Contributor) / School of International Letters and Cultures (Contributor)
Created2021-12
Description
Traumatic brain injury (TBI)—sudden impact or acceleration trauma to the head—is a major cause of death and disability worldwide and is particularly amplified in pediatric cases. TBI is the leading cause of mortality and morbidity in children and adolescents. Adolescence is a critical time where the brain undergoes cognitive development and brain injury-induced disruptions to these processes can lead to life-long debilitating morbidities. The aim of this study was to determine if exercising spatial and contextual memory circuits using a novel rehabilitation strategy called Peg Forest Rehabilitation (PFR) could mitigate the onset of injury-induced cognitive deficits in juvenile rats subjected to diffuse TBI. The PFR aims to synthesize neuroplasticity-based enrichment to improve cognitive outcomes after TBI. We hypothesized that PFR treatment would mitigate the onset of brain injury-induced cognitive deficits and reduce neuroinflammation. Juvenile male Sprague-Dawley rats (post-natal day 35) were subjected to diffuse traumatic brain injury via midline fluid percussion injury or a control surgery. One-week post-injury, rats were exposed to PFR or cage control exploration (15 min/day). PFR allowed free navigation through random configuration of the peg-filled arena for 10 days over 2 weeks. Control rats remained in home cages in the center of the arena with the peg-board removed for 15 min/day/10 days. One-week post-rehabilitation (one-month post-injury), cognitive performance was assessed for short-term (novel object recognition; NOR), long-term (novel location recognition; NLR), and working (temporal order recognition; TOR) memory performance, calculated as a discrimination index between novel and familiar objects. Tissue was collected for immunohistochemistry and stained for ionized calcium binding proteins (Iba-1) to visualize microglia morphology, and somatostatin. PFR attenuated TBI-induced deficits on the NOR task, where the TBI-PFR treatment group spent significantly more time with the novel object compared with the familiar (*p=0.0046). Regardless of rehabilitation, brain-injured rats had hyper-ramified microglia in the hypothalamus indicated by longer branch lengths and more endpoints per cell compared with uninjured shams. Analysis of somatostatin data is ongoing. In this study, passive, intermittent PFR that involved dynamic, novel spatial navigation, prevented TBI-induced cognitive impairment in adolescent rats. Spatial navigation training may have clinical efficacy and should be further investigated.
ContributorsAftab, Umar (Author) / Rowe, Rachel K. (Thesis director) / Newbern, Jason M. (Thesis director) / Ortiz, J. Bryce (Committee member) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2020-05