Matching Items (72)
Description
Random peptide microarrays are a powerful tool for both the treatment and diagnostics of infectious diseases. On the treatment side, selected random peptides on the microarray have either binding or lytic potency against certain pathogens cells, thus they can be synthesized into new antimicrobial agents, denoted as synbodies (synthetic antibodies). On the diagnostic side, serum containing specific infection-related antibodies create unique and distinct "pathogen-immunosignatures" on the random peptide microarray distinct from the healthy control serum, and this different mode of binding can be used as a more precise measurement than traditional ELISA tests. My thesis project is separated into these two parts: the first part falls into the treatment side and the second one focuses on the diagnostic side. My first chapter shows that a substitution amino acid peptide library helps to improve the activity of a recently reported synthetic antimicrobial peptide selected by the random peptide microarray. By substituting one or two amino acids of the original lead peptide, the new substitutes show changed hemolytic effects against mouse red blood cells and changed potency against two pathogens: Staphylococcus aureus and Pseudomonas aeruginosa. Two new substitutes are then combined together to form the synbody, which shows a significantly antimicrobial potency against Staphylococcus aureus (<0.5uM). In the second chapter, I explore the possibility of using the 10K Ver.2 random peptide microarray to monitor the humoral immune response of dengue. Over 2.5 billion people (40% of the world's population) live in dengue transmitting areas. However, currently there is no efficient dengue treatment or vaccine. Here, with limited dengue patient serum samples, we show that the immunosignature has the potential to not only distinguish the dengue infection from non-infected people, but also the primary dengue infection from the secondary dengue infections, dengue infection from West Nile Virus (WNV) infection, and even between different dengue serotypes. By further bioinformatic analysis, we demonstrate that the significant peptides selected to distinguish dengue infected and normal samples may indicate the epitopes responsible for the immune response.
ContributorsWang, Xiao (Author) / Johnston, Stephen Albert (Thesis advisor) / Blattman, Joseph (Committee member) / Arntzen, Charles (Committee member) / Arizona State University (Publisher)
Created2013
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
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
Cancer remains one of the leading killers throughout the world. Death and disability due to lung cancer in particular accounts for one of the largest global economic burdens a disease presents. The burden on third-world countries is especially large due to the unusually large financial stress that comes from late tumor detection and expensive treatment options. Early detection using inexpensive techniques may relieve much of the burden throughout the world, not just in more developed countries. I examined the immune responses of lung cancer patients using immunosignatures – patterns of reactivity between host serum antibodies and random peptides. Immunosignatures reveal disease-specific patterns that are very reproducible. Immunosignaturing is a chip-based method that has the ability to display the antibody diversity from individual sera sample with low cost. Immunosignaturing is a medical diagnostic test that has many applications in current medical research and in diagnosis. From a previous clinical study, patients diagnosed for lung cancer were tested for their immunosignature vs. healthy non-cancer volunteers. The pattern of reactivity against the random peptides (the ‘immunosignature’) revealed common signals in cancer patients, absent from healthy controls. My study involved the search for common amino acid motifs in the cancer-specific peptides. My search through the hundreds of ‘hits’ revealed certain motifs that were repeated more times than expected by random chance. The amino acids that were the most conserved in each set include tryptophan, aspartic acid, glutamic acid, proline, alanine, serine, and lysine. The most overall conserved amino acid observed between each set was D - aspartic acid. The motifs were short (no more than 5-6 amino acids in a row), but the total number of motifs I identified was large enough to assure significance. I utilized Excel to organize the large peptide sequence libraries, then CLUSTALW to cluster similar-sequence peptides, then GLAM2 to find common themes in groups of peptides. In so doing, I found sequences that were also present in translated cancer expression libraries (RNA) that matched my motifs, suggesting that immunosignatures can find cancer-specific antigens that can be both diagnostic and potentially therapeutic.
ContributorsShiehzadegan, Shima (Author) / Johnston, Stephen (Thesis director) / Stafford, Phillip (Committee member) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2015-12
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
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
The influenza virus, also known as "the flu", is an infectious disease that has constantly affected the health of humanity. There is currently no known cure for Influenza. The Center for Innovations in Medicine at the Biodesign Institute located on campus at Arizona State University has been developing synbodies as a possible Influenza therapeutic. Specifically, at CIM, we have attempted to design these initial synbodies to target the entire Influenza virus and preliminary data leads us to believe that these synbodies target Nucleoprotein (NP). Given that the synbody targets NP, the penetration of cells via synbody should also occur. Then by Western Blot analysis we evaluated for the diminution of NP level in treated cells versus untreated cells. The focus of my honors thesis is to explore how synthetic antibodies can potentially inhibit replication of the Influenza (H1N1) A/Puerto Rico/8/34 strain so that a therapeutic can be developed. A high affinity synbody for Influenza can be utilized to test for inhibition of Influenza as shown by preliminary data. The 5-5-3819 synthetic antibody's internalization in live cells was visualized with Madin-Darby Kidney Cells under a Confocal Microscope. Then by Western Blot analysis we evaluated for the diminution of NP level in treated cells versus untreated cells. Expression of NP over 8 hours time was analyzed via Western Blot Analysis, which showed NP accumulation was retarded in synbody treated cells. The data obtained from my honors thesis and preliminary data provided suggest that the synthetic antibody penetrates live cells and targets NP. The results of my thesis presents valuable information that can be utilized by other researchers so that future experiments can be performed, eventually leading to the creation of a more effective therapeutic for influenza.
ContributorsHayden, Joel James (Author) / Diehnelt, Chris (Thesis director) / Johnston, Stephen (Committee member) / Legutki, Bart (Committee member) / Barrett, The Honors College (Contributor) / Department of Psychology (Contributor) / Department of Chemistry and Biochemistry (Contributor)
Created2014-05