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Effect of Rexinoids on Inducing Effector T Cell Chemotaxis

Description

The retinoid-X receptor (RXR) can form heterodimers with both the retinoic-acid
receptor (RAR) and vitamin D receptor (VDR). The RXR/RAR dimer is activated by ligand all
trans retinoic acid (ATRA), which culminates in gut-specific effector T cell migration. Similarly,

The retinoid-X receptor (RXR) can form heterodimers with both the retinoic-acid
receptor (RAR) and vitamin D receptor (VDR). The RXR/RAR dimer is activated by ligand all
trans retinoic acid (ATRA), which culminates in gut-specific effector T cell migration. Similarly,
the VDR/RXR dimer binds 1,25(OH)2D3 to cause skin-specific effector T cell migration.
Targeted migration is a potent addition to current vaccines, as it would induce activated T cell
trafficking to appropriate areas of the immune system and ensure optimal stimulation (40).
ATRA, while in use clinically, is limited by toxicity and chemical instability. Rexinoids
are stable, synthetically developed ligands specific for the RXR. We have previously shown that
select rexinoids can enhance upregulation of gut tropic CCR9 receptors on effector T cells.
However, it is important to establish whether these cells can actually migrate, to show the
potential of rexinoids as vaccine adjuvants that can cause gut specific T cell migration.
Additionally, since the RXR is a major contributor to VDR-mediated transcription and
epidermotropism (15), it is worth investigating whether these compounds can also function as
adjuvants that promote migration by increasing expression of skin tropic CCR10 receptors on T
cells.
Prior experiments have demonstrated that select rexinoids can induce gut tropic migration
of CD8+ T cells in an in vitro assay and are comparable in effectiveness to ATRA (7). The effect
of rexinoids on CD4+ T cells is unknown however, so the aim of this project was to determine if
rexinoids can cause gut tropic migration in CD4+ T cells to a similar extent. A secondary aim
was to investigate whether varying concentrations in 1,25-Dihydroxyvitamin D3 can be linked to
increasing CCR10 upregulation on Jurkat CD4+ T cells, with the future aim to combine 1,25
Dihydroxyvitamin D3 with rexinoids.
These hypotheses were tested using murine splenocytes for the migration experiment, and
human Jurkat CD4+ T cells for the vitamin D experiment. Migration was assessed using a
Transwell chemotaxis assay. Our findings support the potential of rexinoids as compounds
capable of causing gut-tropic migration in murine CD4+ T cells in vitro, like ATRA. We did not
observe conclusive evidence that vitamin D3 causes upregulated CCR10 expression, but this
experiment must be repeated with a human primary T cell line.

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Date Created
2020-05

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Utilizing Confocal Laser Scanning Microscopy to Visualize Origami Nanostructure Transfection in Primary Immune Cells

Description

An aim of fundamental immunology is quantifying the diversity of the T cell receptor (TCR) repertoire to elucidate the vast recognition by T cells for protection against pathogen and cancer. The utilization of DNA origami nanostructures engineered to capture single

An aim of fundamental immunology is quantifying the diversity of the T cell receptor (TCR) repertoire to elucidate the vast recognition by T cells for protection against pathogen and cancer. The utilization of DNA origami nanostructures engineered to capture single cell paired TCR mRNA sequences has transformed the financial and time requirements of repertoire establishment. To further support this protocol, confocal laser scanning microscopy was implemented following transfection to visualize the stability of the DNA origami within primary immune lymphocytes.

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Date Created
2018-05

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CXCL10-Induced Migration of Triple-Negative Breast Cancer Cells

Description

Inhibitor of growth factor 4 (ING4) is a tumor suppressor of which low expression has been associated with poor patient survival and aggressive tumor progression in breast cancer. ING4 is characterized as a transcription regulator of inflammatory genes. Among the

Inhibitor of growth factor 4 (ING4) is a tumor suppressor of which low expression has been associated with poor patient survival and aggressive tumor progression in breast cancer. ING4 is characterized as a transcription regulator of inflammatory genes. Among the ING4-regulated genes is CXCL10, a chemokine secreted by endothelial cells during normal inflammation response, which induces chemotactic migration of immune cells to the site. High expression of CXCL10 has been implicated in aggressive breast cancer, but the mechanism is not well understood. A potential signaling molecule downstream of Cxcl10 is Janus Kinase 2 (Jak2), a kinase activated in normal immune response. Deregulation of Jak2 is associated with metastasis, immune evasion, and tumor progression in breast cancer. Thus, we hypothesized that the Ing4/Cxcl10/Jak2 axis plays a key role in breast cancer progression. We first investigated whether Cxcl10 affected breast cancer cell migration. We also investigated whether Cxcl10-mediated migration is dependent on ING4 expression levels. We utilized genetically engineered MDAmb231 breast cancer cells with a CRISPR/Cas9 ING4-knockout construct or a viral ING4 overexpression construct. We performed Western blot analysis to confirm Ing4 expression. Cell migration was assessed using Boyden Chamber assay with or without exogenous Cxcl10 treatment. The results showed that in the presence of Cxcl10, ING4-deficient cells had a two-fold increase in migration as compared to the vector controls, suggesting Ing4 inhibits Cxcl10-induced migration. These findings support our hypothesis that ING4-deficient tumor cells have increased migration when Cxcl10 signaling is present in breast cancer. These results implicate Ing4 is a key regulator of a chemokine-induced tumor migration. Our future plan includes evaluation of Jak2 as an intermediate signaling molecule in Cxcl10/Ing4 pathway. Therapeutic implications of these findings are targeting Cxcl10 and/or Jak2 may be effective in treating ING4-deficient aggressive breast cancer.

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Date Created
2019-05

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Cloning and expression of antigen-specific T cell receptors

Description

Cancer poses a significant burden on the global health system and represents a leading cause of death worldwide. For late-stage cancers, the traditional treatments of chemotherapy, radiation, and surgery are not always viable, and they can pose unnecessary health risks

Cancer poses a significant burden on the global health system and represents a leading cause of death worldwide. For late-stage cancers, the traditional treatments of chemotherapy, radiation, and surgery are not always viable, and they can pose unnecessary health risks to the patients. New immunotherapies, such as adoptive cell transfer, are being developed and refined to treat such cancers. T cell immunotherapies in particular, where a patient’s T cell lymphocytes are isolated and amplified to be re-infused into the patient or where human cell lines are engineered to express T cell receptors for the recognition of common cancer antigens, are being expanded on because for some cancers, they could be the only option. Constructing an optimal pipeline for cloning and expression of antigen-specific TCRs has significant bearing on the efficacy of engineered cell lines for ACT. Adoptive T cell transfer, while making great strides, has to overcome a diverse T cell repertoire – cloning and expressing antigen-specific TCRs can mediate this understanding. Having identified the high frequency FluM1-specific TCR sequences in stimulated donor PBMCs, it was hypothesized that the antigen-specific TCR could be reconstructed via Gateway cloning methods and tested for expression and functionality. Establishing this pipeline would confirm an ability to properly pair and express the heterodimeric chains. In the context of downstream applications, neoantigens would be used to stimulate T cells, the α and β chains would be paired via single-cell or bulk methods, and instead of Gateway cloning, the CDR3 hypervariable regions α and β chains alone would be co-expressed using Golden Gate assembly methods.

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Date Created
2019-05

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DNA Origami as Novel Immune Adjuvants

Description

Cytokines induced by inflammasome has been used for blood cancer treatments, yet these treatments have been less successful in the solid tumor microenvironment. Here precise-morphology DNA origami structures were implemented to accurately test the effect and mechanism of activation in

Cytokines induced by inflammasome has been used for blood cancer treatments, yet these treatments have been less successful in the solid tumor microenvironment. Here precise-morphology DNA origami structures were implemented to accurately test the effect and mechanism of activation in the NLRP3 inflammasome. THP1 WT cells, a macrophage cell line, were treated with eleven different DNA origami structures. The inflammasome activation of two cytokines, Interleukin 1 beta (IL-1β) and Interferon beta (IFN-β), was measured using HEK Blue IL-1β cells, HEK Blue IFN-β cells, and enzyme linked immunosorbent assay (ELISA). Differences in activation signaling have the potential to provide the characterization required to address the intrinsic complexity of modulating an immune response. It is hoped that DNA origami will help induce more inflammation for solid tumors. The DNA origami was tested in three different volumes: 1 μL, 5 μL, and 10 μL. Overall, the origami that showed promising results were Mg Square. Tetrahedral and P53 block also showed potential but not as well as Mg square. Further testing of more DNA origami structures and testing them in mice are key to the success of targeted cancer immunotherapies in the neoadjuvant setting.

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Date Created
2019-05

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Effects of LCMV Infection on Murine Fetal Development in Immunized Mothers

Description

Despite a continuously growing body of evidence that they are one of the major causes of pregnancy loss, preterm birth, pregnancy complications, and developmental abnormalities leading to high rates of morbidity and mortality, viruses are often overlooked and underestimated as

Despite a continuously growing body of evidence that they are one of the major causes of pregnancy loss, preterm birth, pregnancy complications, and developmental abnormalities leading to high rates of morbidity and mortality, viruses are often overlooked and underestimated as teratogens. The Zika virus epidemic beginning in Brazil in 2015 brought teratogenic viruses into the spotlight for the public health community and popular media, and its infamy may bring about positive motivation and funding for novel treatments and vaccination strategies against it and a variety of other viruses that can lead to severe congenital disease. Lymphocytic choriomeningitis virus (LCMV) is famous in the biomedical community for its historic and continued utility in mouse models of the human immune system, but it is rarely a source of clinical concern in terms of its teratogenic risk to humans, despite its ability to cause consistently severe ocular and neurological abnormalities in cases of congenital infection. Possibilities for a safe and effective LCMV vaccine remain difficult, as the robust immune response typical to LCMV can be either efficiently protective or lethally pathological based on relatively small changes in the host type, viral strain, viral dose, method of infection/immunization, or molecular characteristics of synthetic vaccination. Introducing the immunologically unique state of pregnancy and fetal development to the mix adds complexity to the process. This thesis consists of a literature review of teratogenic viruses as a whole, of LCMV and its complications during pregnancy, of LCMV immunopathology, and of current understanding of vaccination against LCMV and against other teratogenic viruses, as well as a hypothetical experimental design intended to initially bridge the gaps between LCMV vaccinology and LCMV teratogenicity by bringing a vaccine study of LCMV into the context of viral challenge during pregnancy.

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Date Created
2020-05

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Inhibition of PKR phosphorylation by Vaccinia Virus' E3 Protein

Description

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

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.

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Date Created
2017-05

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Does an extended washout period of six weeks following the end of chronic stress continue the benefits on spatial learning and memory?

Description

Chronic stress often leads to cognitive deficits, especially within the spatial memory domain mediated by the hippocampus. When chronic stress ends and a no-stress period ensues (i.e., washout, WO), spatial ability improves, which can be better than non-stressed controls (CON).

Chronic stress often leads to cognitive deficits, especially within the spatial memory domain mediated by the hippocampus. When chronic stress ends and a no-stress period ensues (i.e., washout, WO), spatial ability improves, which can be better than non-stressed controls (CON). The WO period is often the same duration as the chronic stress paradigm. Given the potential benefit of a post-stress WO period on cognition, it is important to investigate whether this potential benefit of a post-stress WO period has long-lasting effects. In this project, chronic restraint (6hr/d/21d) in Sprague-Dawley rats was used, as it is the minimum duration necessary to observe spatial memory deficits. Two durations of post-stress WO were used following the end of chronic restraint, 3 weeks (STR-WO3) and 6 weeks (STR-WO6). Immediately after chronic stress (STR-IMM) or the WO periods, rats were tested on various cognitive tests. We corroborated past studies that chronic stress impaired spatial memory (STR-IMM vs CON). Interestingly, STR-WO3 and STR-WO6 failed to demonstrate improved spatial memory on a radial arm water maze task, performing similarly as STR-IMM. Performance outcomes were unlikely from differences in anxiety or motivation because rats from all conditions performed similarly on an open field task and on a simple object recognition paradigm, respectively. However, performance on object placement was unusual in that very few rats explored, suggesting some degree of anxiety or fear in all groups. One possible interpretation of the unusual results of the 3 week washout group may be attributed to the different spatial memory tasks used across studies or external factors from the study. Further exploration of these other factors led to the conclusion that they did not play a role and the STR-WO3 RAWM data were anomalous to other studies. This suggests that a washout period following chronic stress may not be fully understood.

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2017-05

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Analysis for antitumor antibody signatures in lung cancer using two different random peptide libraries

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

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.

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Date Created
2015-12

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The Role of Tumor Necrosis Factor Receptors p55 and p75 in Acute IL-2 Toxicity in Chronic Viral Infections

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

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.

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Date Created
2014-05