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Since Metastatic Osteosarcoma is unresponsive to most of the current standards of care currently available, and yields a survival rate of 20%, it is pertinent that novel approaches to treating it be undertaken in scientific research. Past studies in our lab have used a The Immune Blockade Therapy, utilizing α-CTLA-4 and α-PD-L1 to treat mice with metastatic osteosarcoma; this resulted in 60% of mice achieving disease-free survival and protective immunity against metastatic osteosarcoma. 12 We originally wanted to see if the survival rate could be boosted by pairing the immune blockade therapy with another current, standard of care, radiation. We had found that there were certain, key features to experimental design that had to be maintained and explored further in order to raise survival rates, ultimately with the goal of reestablishing the 60% survival rate seen in mice treated with the immune blockade therapy. Our results show that mice with mature immune systems, which develop by 6-8 weeks, should be used in experiments testing an immune blockade, or other forms of immunotherapy, as they are capable of properly responding to treatment. Treatment as early as one day after should be maintained in future experiments looking at the immune blockade therapy for the treatment of metastatic osteosarcoma in mice. The immune blockade therapy, using α-PD-L1 and α-CTLA-4, seems to work synergistically with radiation, a current standard of care. The combination of these therapies could potentially boost the 60% survival rate, as previously seen in mice treated with α-PD-L1 and α-CTLA-4, to a higher percent by means of reducing tumor burden and prolonging length of life in metastatic osteosarcoma.
ContributorsLabban, Nicole (Author) / Blattman, Joseph (Thesis director) / Appel, Nicole (Committee member) / Barrett, The Honors College (Contributor)
Created2017-05
Description
Memory CD8+ T-cells can persist in the absence of antigen, primed for immediate activation and proliferation if later exposed to the same antigen. These cytotoxic lymphocytes provide long-term immunity following an acute infection. Studies have observed that intermediate levels of general T cell transfer prior to infection may cause an inappropriate response resulting in increased pathology rather than prevention. Therefore, our study focused on a memory CD8 T-cell therapy using lymphocytic choriomeningitis virus (LCMV) specific splenocytes, which activate and proliferate at an accelerated pace compared to that of naive T-cells. LCMV is a natural murine pathogen which also poses a zoonotic infection threat to humans, and the effect of immune cell vaccination therapies for LCMV is not fully understood. We observed the effect of multiple memory CD8 T cell dosage levels on overall disease and memory CD8 T-cell response to the virus. Infection by exposure to a carrier was shown to have a reduced impact on mice receiving higher doses of memory T cells prior to infection compared to mice receiving less or no memory cells. Higher presence of activated memory cells were shown to correlate with less disease-related weight loss and accelerated recovery times. Survival rate after exposure to carriers was not shown to be affected by dosage level, warranting further research regarding the prevalence of the immunopathology observed in other studies in natural murine transmission models.
ContributorsMiller, Charles (Author) / Blattman, Joseph (Thesis director) / Holechek, Susan (Committee member) / Carmen, Joshua (Committee member) / W. P. Carey School of Business (Contributor) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2016-05
Description
Non-small cell lung cancer (NSCLC) has become the leading cause of cancer-related deaths in the United States with a combined 5-year survival rate of only 16%. Even with advancements in aggressive chemotherapeutics, there has been little improvement in patient survival. LKB1 (liver kinase B1)/STK11 (serine-threonine kinase 11) is a tumor suppressor gene mutated in ~30% of NSCLC adenocarcinomas and loss of LKB1 is associated with a more aggressive cancer phenotype. In LKB1-deficient NSCLC, we observe significantly elevated expression and secretion of the chemokines CCL2, CCL5, and CCL20, which are involved in macrophage recruitment. Numerous studies have shown that high infiltration of a unique subset of macrophages called tumor-associated macrophages (TAMs) is associated with poor prognosis in patients with various cancers. mTORC1-HIF1-α and NFκB are two pathways that have been shown to regulate chemokine secretion and are often up-regulated in the absence of LKB1. Dosing LKB1-null cell lines with inhibitors of mTOR and NFκB in addition to silencing HIF1-α gene expression demonstrate that NFκB but not mTORC1-HIF1-α signaling may play a role in regulating chemokine secretion in LKB1-deficient NSCLC. Collectively, these results provide insight into the mechanisms responsible for the aggressive phenotype associated with LKB1-deficient non-small cell lung cancer.
ContributorsO'Brien, Kelley Xiao-Fung (Author) / Blattman, Joseph (Thesis director) / Inge, Landon (Committee member) / Friel, Jacqueline (Committee member) / Barrett, The Honors College (Contributor) / Department of Psychology (Contributor) / School of Life Sciences (Contributor)
Created2015-05
Description
Among wild rodent populations, vertical transmission is believed to constitute the primary route of infection for Lymphocytic Choriomeningitis Virus (LCMV), a non-lytic arenavirus with both acute and chronic forms. When carrier mice infected at birth with the acute Armstrong strain reproduce, they generate congenital carrier offspring containing a quasispecies of LCMV that includes Armstrong as well as its chronic Clone-13 variant. This study examined the genetic trends in the vertical transmission of LCMV from mothers infected perinatally with Clone-13. Viral isolates obtained from the serum of congenital carrier offspring were partially sequenced to reveal residue 260 in the glycoprotein-encoding region of their S segment, the site of a major amino acid change differentiating the chronic and acute strains. It was found that the phenylalanine-to-leucine mutation associated with Clone-13 was present in 100% of the isolates, strongly indicating that the offspring of Clone-13 carriers contain exclusively the chronic variant. This research has broad implications for the epidemiology of the virus, and, given the predominance of Armstrong in the wild, suggests that there must be a biological cost associated with Clone-13 infection in non-carriers.
ContributorsFrear, Cody Christian (Author) / Blattman, Joseph (Thesis director) / Hogue, Brenda (Committee member) / Holechek, Susan (Committee member) / Barrett, The Honors College (Contributor) / School of Human Evolution and Social Change (Contributor) / School of Life Sciences (Contributor)
Created2015-05
Description
Efforts to quantify the diversity of the T cell repertoire have generally been unsuccessful because not all factors accounting for diversity have been considered. In order to get an accurate representation of the T cell repertoire, one must incorporate analysis of germline gene diversity, diversity from somatic recombination, joining diversity from N- and P- nucleotides, and TCR chain pairing diversity. Because of advances in high-throughput sequencing techniques, estimates have been able to account for diversity from TCR genes. However the ability to account for chain pairing diversity has been more difficult. In order to do so, single cell sorting techniques must be employed. These techniques, though effective, are time consuming and expensive. For this reason, no large-scale analyses have been done on the immune repertoires using these techniques. In this study, we propose a novel method for linking the two TCR chain sequences from an individual cell. DNA origami nanostructure technology is employed to capture and bind the TCRγ and TCRδ chain mRNA inside individual cells using probe strands complementary to the C-region of those sequences. We then use a dual-primer RT and ligation molecular strategy to link the two sequences together. The result is a single amplicon containing the CDR3 region of the TCRγ and TCRδ. This amplicon can then be easily PCR amplified using sequence specific primers, and sequenced. DNA origami nanostructures offer a rapid, cost-effective method alternative to conventional single cell sorting techniques, as both TCR mRNA can be captured on one origami molecule inside a single cell. At present, this study outlines a proof-of-principle analysis of the method to determine its functionality. Using known TCRγ and TCRδ sequences, the DNA origami and RT/PCR method was tested and resulting sequence data proved the effectiveness of the method. The original TCRγ and TCRδ sequences were linked together as a single amplicon containing both CDR3 regions of the genes. Thus, this method can be employed in further research to elucidate the γδ T cell repertoire. This technology is also easily adapted to any gene target or cell type and therefore presents a large opportunity to be used in other immune repertoire analysis and other immunological studies (such as the rapid identification and subsequent production of antibodies).
ContributorsPoindexter, Morgan Elizabeth (Author) / Blattman, Joseph (Thesis director) / Yan, Hao (Committee member) / Schoettle, Louis (Committee member) / Barrett, The Honors College (Contributor) / School of Life Sciences (Contributor)
Created2015-05
Description
Patients who are infected with the human immunodeficiency virus (HIV) and who remain adherent to their highly active antiretroviral treatment (HAART) regimen are likely to achieve good virologic control over significant periods of time. Children who start with a low CD4 percentage (below 15%) are associated with adverse clinical outcomes and the risk of never increasing their CD4 counts to normal functioning levels. While adherent adult HIV patients have been studied frequently, this was a retrospective chart study that aimed to describe the immune reconstitution pattern for up to 16 years in virologically controlled pediatric patients who had been or who are currently being treated at the Bill Holt Clinic at Phoenix Children's Hospital. In the preliminary study, 35 patients met criteria for inclusion and three years later for this extension study 7 more were added while 17 of the initial patients were followed further because they have remained in care and virologically controlled. All 28 patients who achieved 5 years of viral suppression were >25% CD4. All 8 patients who achieved 12 years of viral suppression were >31% CD4. All patients who achieved 16 years of viral suppression were >41% CD4. After 12 years, the 8 patients who maintained viral suppression all had absolute CD4 counts of over 600 cells and additionally each had CD4/CD8 ratios greater than 1. Overall, the data shows immune system normalization for up to 16 years, although CD4/CD8 ratios improved but never completely normalized. Some limitations include a small sample size and missing data points due to laboratory testing errors or the lack of technology in different countries to test for CD8 cells. These findings suggest that children who remain adherent to HAART can experience ongoing immune healing for up to 16 years. This may provide additional incentive to providers and caretakers to encourage adherence and maximize long-term immune competence in HIV positive children.
ContributorsRichards, Anne Elizabeth (Author) / Wachter, Rebekka (Thesis director) / Blattman, Joseph (Committee member) / Barrett, The Honors College (Contributor) / Department of Chemistry and Biochemistry (Contributor) / School of Human Evolution and Social Change (Contributor)
Created2015-05
Description
Glioblastoma multiforme (GBM) is an aggressive malignant brain tumor with a median prognosis of 14 months. Human hairless protein (HR) is a 130 kDa nuclear transcription factor that plays a critical role in skin and hair function but was found to be highly expressed in neural tissue as well. The expression of HR in GBM tumor cells is significantly decreased compared to the normal brain tissue and low levels of HR expression is associated with shortened patient survival. We have recently reported that HR is a DNA binding phosphoprotein, which binds to p53 protein and p53 responsive element (p53RE) in vitro and in intact cells. We hypothesized that HR can regulate p53 downstream target genes, and consequently affects cellular function and activity. To test the hypothesis, we overexpressed HR in normal human embryonic kidney HEK293 and GBM U87MG cell lines and characterized these cells by analyzing p53 target gene expression, viability, cell-cycle arrest, and apoptosis. The results revealed that the overexpressed HR not only regulates p53-mediated target gene expression, but also significantly inhibit cell viability, induced early apoptosis, and G2/M cell cycle arrest in U87MG cells, compared to mock groups. Translating the knowledge gained from this research on the connections between HR and GBM could aid in identifying novel therapies to circumvent GBM progression or improve clinical outcome.
ContributorsBrook, Lemlem Addis (Author) / Blattman, Joseph (Thesis director) / Hsieh, Jui-Cheng (Committee member) / Goldstein, Elliott (Committee member) / Harrington Bioengineering Program (Contributor) / School of Social Transformation (Contributor) / Barrett, The Honors College (Contributor)
Created2018-05
Description
Viral infections are a significant cause of disease in humans. While some viral diseases have been eliminated, many more continue to infect millions. Viral infections are challenging to treat because viruses use host cell machinery to replicate, so it is difficult to develop drugs that can target viruses. Normally, the host’s immune system is capable of destroying the virus, but during chronic infections it becomes exhausted and T cells lose their effector functions necessary for the clearance of the virus. IL-2 can help relieve this exhaustion, but causes toxicity to the body. In mice infected with chronic LCMV, IL-2 administration causes death due to pulmonary hemorrhage. CD4 deficient mice were infected with chronic LCMV and then dosed with IL-2 and survived, but mice that were deficient for CD8 T cells died, indicating that toxicity was mediated by CD8 T cells. CD8 T cells can kill infected host cells directly by producing perforin, or can produce cytokines like IFN-γ and TNF to further activate the immune system and mediate killing. Mice that were deficient in perforin died after IL-2 administration, as well as mice that were deficient in IFN-γ. Mice deficient in TNF, however, survived, indicating that TNF was mediating the toxicity in response to IL-2. There are two different receptors for TNF, p55 and p75. p55 is known as TNFR1 and has been implicated in apoptosis of virally infected cells. P75 is known as TNFR2 and is associated more with inflammation in response to infection. My hypothesis was that if TNFR2 was knocked out, infected mice would survive IL-2 dosing. When single knockouts of TNFR1 and 2 were used in an experiment however, it was found that either receptor is capable of mediating toxicity, as both experimental groups failed to survive. This is relevant to current IL-2 therapies because there is no way to eliminate a single receptor in order to reduce toxicity. Further studies exploring the anti-viral capabilities of IFN-γ are suggested.
ContributorsJarvis, Jordan Alisa (Author) / Blattman, Joseph (Thesis director) / Denzler, Karen (Committee member) / McAfee, Megan (Committee member) / Barrett, The Honors College (Contributor) / School of Life Sciences (Contributor)
Created2014-05
Description
Pathogens such as lymphocytic choriomeningitis virus (LCMV) cause abnormalities in the nervous system of developing mice and humans. While humans are able to recover from infection and clear the virus, the mouse immune system tolerates the virus and lifelong infection ensues. In order to understand the factors driving LCMV evolution and evaluate its neuropathogenesis, a mouse model was needed. To establish congenital infection, newborn C57BL/6J mice were intra-cerebrally (i.c.) injected with 1 x 103 PFU LCMV Armstrong. Mice failed to thrive, resulting in a linear reduction in survival over the following two weeks and overall survival of 13%. Surviving mice did not have virus in their circulation after thirty days. As an alternative, 500 PFU of LCMV Armstrong was injected intraperitoneally (i.p.) into other litters. While this was associated with significantly reduced mortality, no mice in this group developed persistent infection either. ELISAs revealed that the mothers of injected pups developed a robust humoral response, confirming earlier reports that contact-associated acute infection occurs (Hotchin, 1971). In addition, the offspring of two litters of mice (out of six tested) also had antibodies to the virus, but at slightly lower titers. This indicates that the humoral response of the mothers may play a role in the neonatal clearance of infection. A higher titer of LCMV in i.p. injections may be necessary to overcome these barriers and establish chronic infection. In contrast, a lower dose of LCMV is recommended for i.c. injections, as the mortality seemed directly linked to the effects of the virus on offspring growth and development. Exposure to the virus in utero may also be necessary to increase survival and the likelihood of chronic infection.
ContributorsMorrow, Kristen Nicole (Author) / Blattman, Joseph (Thesis director) / Holechek, Susan (Committee member) / Franco, Lina (Committee member) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2016-05
Description
Human Immunodeficiency Virus type 1 (HIV-1) causes millions of deaths every year, but a protective vaccine remains elusive. A promising vaccine strategy is to use virus-like particles (VLPs) for HIV-1. To this end, HIV-1 VLPs were produced in Nicotiana benthamiana plants that were stably expressing the HIV-1 Gag protein and transiently expressing a truncated form of gp41. These VLPs were tested to determine their inherent adjuvant effects due to their production in plants in order to dissect the previously observed stimulating activity of these VLPs in a prime-boost vaccine approach. THP1 human monocytes were differentiated using PMA or IL-4 and GM-CSF to form macrophages and dendritic cells, respectively. These cells were treated with purified VLPs or control samples to determine the individual adjuvant effects of the plant, bacterial, and VLP components in the purified VLP samples. It was postulated that the PMA-differentiated THP1 cells were not induced to become macrophages due to the lack of CD11b+ cells in the sample and the lack of increased TNFα expression in response to LPS treatment. It was also determined that the VLPs have inherent adjuvant properties to dendritic cells due to bacterial and VLP components, but not due to plant components.
ContributorsDickey, Rebekah Marie (Author) / Mor, Tsafrir (Thesis director) / Blattman, Joseph (Committee member) / Meador, Lydia (Committee member) / Barrett, The Honors College (Contributor) / School of Life Sciences (Contributor)
Created2015-05