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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.
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.
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.
Background: Coccidioidomycosis (Valley Fever) is a respiratory disease that is caused by the soil-dwelling fungi Coccidioides immitis and Coccidioides posadasii. Because fungal glycosylation patterns are distinct from mammalian glycosylation patterns, we hypothesized that certain lectins (carbohydrate-binding proteins) might have differential binding properties to coccidioidal glycoproteins, and therefore serve as a tool for the purification and characterization of these glycoproteins from patient specimens. Materials and Methods: To identify potential Coccidioides-binding lectins, lectin-based immunohistochemistry was performed using a panel of 21 lectins on lung tissue from human patients infected with Coccidioides. Enzyme-Linked Immunosorbent Assays (ELISAs) were used to confirm and test candidate Coccidioides-binding lectins for their ability to bind to proteins from antigen preparations of laboratory-grown Coccidioides. Inhibition IHC and ELISAs were used to confirm binding properties of these lectins. SDS-PAGE and mass spectrometry were performed on eluates from coccidioidal antigen preparations run through lectin-affinity chromatography columns to characterize and identify lectin-binding coccidioidal glycoproteins. Results: Two GlcNAc-binding lectins, GSLII and sWGA, bound specifically to spherules and endospores in infected human lung tissue, and not to adjacent lung tissue. The binding of these lectins to both Coccidioides proteins in lung tissue and to coccidioidal antigen preparations was confirmed to have lectin-like characteristics. SDS-PAGE analysis of eluates from lectin-affinity chromatography demonstrated that GSLII and sWGA bind to coccidioidal glycoproteins. Mass spectrometric identification of the top ten lectin affinity-purified glycoproteins demonstrated that GSLII and sWGA share affinity to a common set of coccidioidal glycoproteins. Conclusion: This is the first report of lectins that bind specifically to Coccidioides spherules and endospores in infected humans. These lectins may have the potential to serve as tools for a better method of detection and diagnosis of Valley Fever.
Purinergic receptors sense extracellular nucleotide DAMPs such as ATP and adenosine, which are expressed in high concentrations in the tumor microenvironment (TME). A2AR, an adenosine receptor that is expressed on both T cells and tumor cells, promotes immunosuppression. However, the impact of the TME on changes in purinergic receptor expression on CD8 T cells, as well as the overall dynamic between A2AR expression and tumor control, have not been clearly elucidated. Using in vitro co-culture experiments and in vivo murine tumor models, we found that A2AR is significantly upregulated on effector, tumor-infiltrating CD8 T cells. This upregulation was independent of the hypoxia, which we identified via inhibition of HIF1A. We found that this upregulation was partially dependent on CD8 T cell-tumor contact, but independent of cognate antigen recognition, by using transwell co-cultures, as well as combinations of different transgenic lines of CD8 T cells and tumor cells. We confirmed this observation in vivo using transfer of activated OTI cells into B16.OVA-bearing mice. Ultimately, we observed that the upregulation depended on inhibitory receptors such as Tim3 via the antibody blockade of Tim3. Using CRISPR/Cas9-mediated knockout of A2AR on activated CD8 T cells, we found that tumor-bearing mice receiving A2AR knockout CD8 T cells had increased tumor control. Taken together, these results suggest that inhibitory receptor-dependent, TCR-independent signals in the TME promotes upregulation of A2AR on CD8 T cells, leading to impairment of CD8 T cell-mediated tumor control.
Tissue regeneration is a complex process that activates both developmental and metabolic signaling pathways (Kashio & Miura, 2020). The wing imaginal disc in Drosophila melanogaster has been invaluable in discerning what pathways are activated during tissue regeneration, which is typically done by genetically or physically wounding the wing disc and using fluorescent markers to track different signals. However, despite its importance in other regeneration contexts (Tafesh-Edwards & Eleftherianos, 2020), immune signaling has not been well studied in this tissue. Furthermore, what we do know about tissue regeneration and immune signaling is specific to apoptotic cellular death, less is known about other types of cellular death, such as necrotic cellular death and the consequent signaling systems that result from necrosis. Drosophila have an open immune system and only possess innate immunity (Pastor-Pareja et al., 2008), making them an ideal model to study hemocyte involvement in tissue regeneration. Hemocytes are equivalent to blood cells in vertebrates, and are involved in immunological response (Kurucz et al., 2003). In this work, we observed hemocyte accumulation during injury-induced regeneration. Cellular damage was induced using a genetic ablation system known as DUAL Control, with hemipterous CA and GluR1 used to induce apoptotic and necrotic cell death respectfully. We have discovered that while hemocytes are recruited to the wing disc upon both apoptotic and necrotic injury, necrotic tissue has more hemocytes adhered than apoptotic tissue. The difference in adherence could be due to basement membrane integrity being damaged more severely in necrotic discs than apoptotic discs. Our results show that hemocytes are attracted to wing discs that have undergone necrotic damage, indicating that the immune system plays some sort of role in necrotic cellular death. Though the immune response to different types of tissue damage in Drosophila is much simpler than in vertebrate models, there are many similarities between the two, and could lead to research involving human immune signaling as it pertains to regeneration.
The study of broad therapeutic advantages of dance is a growing field of interdisciplinary study. Yet, direct health benefits of dance from a molecular standpoint are still largely unknown. Literature review of dance performance displays in birds as well as other creatures and use of creative tools to analyze the diverse, lifelong experiences of dancers helped shed some light on the subject. Although dance experience exposes harms tied to the social constraints of how the form is experiences buried under joyful takeaways of dance, research supports overall health benefits from moderate amounts of dance maintained in perfect equilibrium.
For my thesis, Professor Florsheim and I decided to focus on building lab experience in preparation for my master’s thesis. This included reading various research papers, starting a mast cell culture, and learning techniques essential for lab work. Additionally, I would conduct presentations and work with my peers to learn about various testing methods and components of the lab. One of the most crucial components of this experience included learning about how to collect bone marrow mast cells including how to properly sacrifice a mouse. My final product is the grant proposal which is what my focus will be for my master’s year.
The purpose of this study was to examine the effects of two positive discrete emotions, awe and nurturant love, on implicit prejudices. After completing an emotion induction task, participants completed Implicit Association Test blocks where they paired photos of Arab and White individuals with "good" and "bad" evaluations. We hypothesized that nurturant love would increase the strength of negative evaluations of Arab individuals and positive evaluations of White individuals, whereas awe would decrease the strength of these negative evaluations when compared to a neutral condition. However, we found that both awe and nurturant love increased negative implicit prejudices toward Arab individuals when compared to the neutral condition.
Vaccinia virus is a cytoplasmic, double-stranded DNA orthopoxvirus. Unlike mammalian cells, vaccinia virus produces double-stranded RNA (dsRNA) during its viral life cycle. The protein kinase R, PKR, is one of the principal host defense mechanisms against orthopoxvirus infection. PKR can bind double-stranded RNA and phosphorylate eukaryotic translation initiation factor, eIF2α, shutting down protein synthesis and halting the viral life cycle. To combat host defenses, vaccinia virus encodes E3, a potent inhibitor of the cellular anti-viral eIF2α kinase, PKR. The E3 protein contains a C-terminal dsRNA-binding motif that sequesters dsRNA and inhibits PKR activation. We demonstrate that E3 also interacts with PKR by co-immunoprecipitation. This interaction is independent of the presence of dsRNA and dsRNA-binding by E3, indicating that the interaction is not due to dsRNA-bridging.
PKR interaction mapped to a region within the dsRNA-binding domain of E3 and overlapped with sequences in the C-terminus of this domain that are necessary for binding to dsRNA. Point mutants of E3 were generated and screened for PKR inhibition and direct interaction. Analysis of these mutants demonstrates that dsRNA-binding but not PKR interaction plays a critical role in the broad host range of VACV. Nonetheless, full inhibition of PKR in cells in culture requires both dsRNA-binding and PKR interaction. Because E3 is highly conserved among orthopoxviruses, understanding the mechanisms that E3 uses to inhibit PKR can give insight into host range pathogenesis of dsRNA producing viruses.