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2017 Research Showcase
IVCB Abstracts
Abstract Title : Role of cytotoxic T cells in the development of non-alcoholic steatohepatitis related fibrosis and progression to hepatocellular carcinoma
Abstract : Hepatocellular carcinoma (HCC) is one of the most prevalent malignancies worldwide and a leading cause of cancer related deaths. Despite major gains in fighting hepatitis virus B and C, the epidemic of liver disease continues to grow with clear links to obesity and alcohol abuse. Non-alcoholic steatohepatitis (NASH) is major driver of HCC but the mechanisms underlying the progression to HCC are poorly understood because they depend on research using mouse models. Many animal models do not accurately mimic human liver disease and do not elicit NASH driven HCC. Here, we used clinical markers to define the best mouse model to mimic human NASH and NASH driven HCC. Classical signs of NASH including fibrosis, steatosis, serum and liver IgA, and lymphocyte infiltration were assessed in MUP-uPA, MCD, STAM and CCl4- induced fibrosis models. IgA levels were increased in high fat diet (HFD) fed MUP-uPA mice, STAM and CCl4 models and minimally in MCD mice. Histological characterizations of the models showed extensive fibrosis and steatosis in HFD fed MUP-uPA mice, modest fibrosis in HFD fed STAM mice, and minimal fibrosis but extensive steatosis in MCD fed mice. Only STAM and MUP-uPA developed tumors. The MUP-uPA model was deemed most faithful in mimicking human NASH because it was the only model in which mice development fibrosis, steatosis, ballooning hepatocytes, elevated serum and liver IgA, systematic weight gain, and tumors, with a long latency. As NASH is a disease based on inflammation, bacteria and immune cells, additional immunological knockout mice were analyzed to investigate the role of adaptive immune cells (T and B cells) in NASH driven HCC, which are poorly understood due to lack of suitable mouse models. CD8+ T cell deficiency enhanced liver fibrosis in NASH models and progression to HCC, with faster onset and increased development of tumors than the corresponding MUP-uPA livers.
Abstract Title : IRF7 is essential for collagen induced arthritis in mice
Abstract : Rheumatoid arthritis is a chronic debilitating disease that not only involves abnormal B cell and T cell behavior but also results in activation of the innate immune system. The innate immune response is regulated by a cascade of signaling events including Interferon regulatory factor 7, IRF7, which is a member of the interferon regulatory factor family of transcription factors. To better understand the role of this transcription factor in the development of disease we tested mice with a genetically engineered deficiency in IRF7 (Irf7-/-) for the development and progression of induced arthritis. Wildtype DBA/J and DBA/J Irf7-/- mice were immunized with type II bovine collagen. In one experiment the mice also received a “boost” with lipopolysaccharide (LPS) to augment the autoantibody response. In the experiment where the mice received the LPS there was significantly more joint swelling in the WT mice. IRF7 is a key regulator in the full penetration of collagen induced arthritis.
Abstract Title : Evaluation of IL-10 in an Animal Model of Rheumatoid Arthritis
Abstract : Rheumatoid arthritis (RA) develops in 1.5 million Americans each year. This is a chronic disabling disease that results in marked joint deformity and disability.  Although antibody blockade has been useful in reducing pro-inflammatory cytokine activity, anti-inflammatory cytokines like interferon beta and IL-10 have not been effective, despite clinical utility in other autoimmune diseases such as multiple sclerosis. We set up a system to examine this disparity using the K/BxN serum transfer model of murine RA. In this model we found there was an increase in the paw swelling in IL10 deficient mice, but the swelling was not completely abrogated. In addition there was a dose response to administering IL-10 exogenously, which was incomplete and plateaued at 20µg/kg.  This system will be used to evaluate other agents for the induction of IL-10 as the mechanism of action to optimize future therapies.
Abstract Title : Chronic E-Cigarette Vapor Inhalation Induces Hepatic Fibrosis in Mice
Abstract : The ever-growing prevalence of the e-cigarette/vaping community signifies the importance of understanding their effects on public health. While often argued that e-cigarettes are safer than traditional tobacco use, there is still extensive research that needs to be done in order to evaluate both the benefits and deleterious effects of chronic e-cigarette vapor (EV) inhalation. We hypothesized that daily e-cigarette use would cause stress to the lung, and the release of stress-signals into systemic circulation. The liver is often one of the first organs affected by circulating pro-fibrotic factors, therefore we designed these studies to evaluate potential effects of EV inhalation on the hepatic system. 6-8 week old outbred CD-1 mice were exposed to one hour of EV daily, via nose-only restraints as part of the inExpose system (SciReq) for 6 months. Livers were harvested and placed into 10% formalin for 48 hours, followed by paraffin embedding, sectioning and Masson Trichrome staining. Blinded grading of the degree of fibrosis in Air controls (restrained by only inhaled room air) versus EV exposed mice was done by taking an average of 15 images per liver. ImageJ software was utilized. First, the desired image was converted to an RGB stack. Next, a montage was made, separating the RGB stack into the 3 different colors for analysis. Since blue dye identifies collagen deposition, a marker of fibrosis, the blue channel was selected for analysis. Once the image was prepared, threshold values were set to a low of 55 and a high of 85, which was the standard used across all images. Using the draw tool we quantified the area of each vessel. Finally, the area was measured for percent value of stained tissue, allowing us to gather data on fibrosis levels in various sized blood vessels. The analysis described an overall significant increase of 9.05% in Trichrome staining, indicative of higher levels of fibrosis in livers from EV exposed mice (p < 0.0001 by two-tailed t-test; Figure 1). Nicotine containing e-liquid, when vaped through a commercially available system, led to hepatic fibrosis in a mouse inhalation model. These data suggest that chronic, daily e-cigarette vapor inhalation may pose a threat to distal organs in human users, and may increase the risk of hepatic dysfunction and disease. ADDITIONAL PRESENTERS: Ashley Du, Albert Tran, Alex Mohensky.
Advisor : ANN FEENEY
Abstract Title : The Orientation of Cer Shapes Kappa Light Chain Repertoire in B Cells
Abstract : B cells are players in the adaptive immune system and a diverse repertoire of B cell receptors is needed for recognizing and fighting off different foreign antigens. Recombination of immunoglobulin gene segments, V(D)J rearrangement, is essential in generating such a diverse B cell repertoire. Our study focuses on rearrangement of V and J segments of Igk light chain locus, the preferentially used light chain locus in mice which consists of ~100 functional Vk genes and four Jk genes. This complicated rearrangement process is facilitated by locus contraction which gives Vk genes throughout the Igk locus a chance to come into proximity with the Jk genes to be recombined at the pre-B cell stage. Studies show that the 650bp V-J intervening ”Contracting element for recombination” (Cer) genomic element is important for locus contraction. Germline deletion of Cer in mice reduces Igk locus compaction and markedly increased rearrangement of Jk-proximal Vk genes and decreased rearrangement of Jk-distal Vk genes. Two CTCF sites oriented toward Vk genes have been identified within Cer. CTCF is known to facilitate long-range interactions, and these interactions usually occur between CTCF sites in convergent orientations. A few convergent CTCF sites are found in the Jk -proximal Vk region but most are located in the Jk distal Vk region. Our goal was to study if orientation of Cer has an effect on rearrangement of Vk genes and long range looping. To do this, we deleted and inverted the Cer element in 445 RAG-/- Abl cell line using CRISPR/Cas9 genome editing. Igk rearrangement was assessed by treating cells with Gleevac tyrosine kinase inhibitor which promotes pre-B cell development in the cell line. We found that inversion of Cer had a similar effect on rearrangement as deletion of Cer, in that both greatly increased rearrangement of Jk-proximal Vk genes. Both Cer deletion and inversion displayed reduced rearrangement of Jk -distal Vk genes, although the effect was more profound in deletion clones. Our data suggests that the orientation of Cer is critical to shape the Vk repertoire.
Advisor : WEG M. ONGKEKO
Abstract Title : RNA-sequencing reveals HPV-specific dysregulation of PIWI-interacting RNAs in head and neck squamous cell carcinoma
Abstract : Human papillomavirus (HPV) is the fastest growing cause of head and neck squamous cell carcinoma (HNSCC), the sixth deathliest cancer worldwide. With the recent emergence of non-coding RNAs as mediators of HNSCC, we decided to investigate HPV-specific alterations of PIWI-interacting RNAs (piRNAs), the largest class of non-coding RNAs that have roles in heterochromatin modification, silencing of retrotransposons, and germ cell maintenance. Using RNA-sequencing data from 466 HNSCC patients in The Cancer Genome Atlas (TCGA), we identified 39 piRNAs significantly dysregulated in HNSCC HPV16(+) vs. normal HPV(-) cohorts (FDR < 0.05). We correlated these piRNAs with clinical variables, recognizing 6 transcripts, most notably NONHSAT077364, NONHSAT108298, and NONHSAT069719, for their associations with tumor stage, metastatic factors, survival, and/or anatomic site. We then analyzed mRNA expression data for the same 466 patients from TCGA and found PIWIL2, a protein known to functionally interact with piRNAs, to be dysregulated in HPV-related HNSCCs and to associate with expression levels of HPV-dysregulated piRNAs (p < 0.05). We subsequently validated the dysregulation of all significant piRNA and PIWI protein candidates in vitro in normal oral epithelial cells ectopically expressing HPV E6/E7. Collectively, our findings reveal the potential of these transcripts as diagnostic or prognostic biomarkers of HNSCC and provide novel insights to thwart the progression of this disease. ADDITIONAL PRESENTERS: Aswini Krishnan
Abstract Title : The Non-apoptotic Roles of Caspase-8 in Tumor Cell Malignancy
Abstract : Caspase-8 is an intracellular protease that plays a key role in initiating apoptosis, a type of programmed cell death. Apoptosis is a key mechanism that suppresses the development of cancer, and so it was surprising that elevated levels of caspase-8 were found to be expressed in most cancer cell lines. Knockdown of caspase-8 had no effect on cell survival in normal cell culture, but in 3-dimensional agar, caspase-8 was detrimental to clonal growth, reinforcing the idea that it suppressed cancer development. The continued expression of caspase-8, in these cells, suggested it might have functions other than apoptosis. Since cells in 2D have a more rigid substrate we tested whether the expression of caspase-8 influenced attachment to that substrate. In short time frames, cells that express caspase-8 adhered slower to tissue culture substrates than cells that were deficient in caspase-8. Immunohistochemistry performed on the cell lines shows that more Focal Adhesion Kinase (FAK) is phosphorylated in cells that expressed caspase-8. Since FAK is a key protein in cell spreading and mobility, this lack of FAK activation may influence both adhesion and, in vivo, metastasis. Therefore, my results support a model in which tumor cells are able to re-purpose, and even depend on, caspase-8 function for malignancy.
Abstract Title : Characterization of imaging changes in brain metastases following combination therapy with SRS and immune checkpoint blockade
Abstract : Abstract Text: Purpose/Objective(s): While stereotactic radiosurgery (SRS) has emerged as the primary treatment modality for brain metastases, emerging evidence suggests that immune checkpoint blockade (ICB) agents have activity in a variety of tumors in the central nervous system (CNS). Furthermore, when delivered concurrently with SRS, ICB may improve intracranial tumor control. While multiple studies have characterized serial imaging characteristics of brain tumors following SRS, there is limited evidence examining post-SRS changes following concurrent SRS and ICB therapy. This study aims to elucidate the use, timing, and effects of the concurrent SBRT and immunotherapy on tumors within the CNS. Materials/Methods: This retrospective study examined serial MRIs of patients with metastatic brain tumors who received concurrent linear accelerator-based SRS and ICB from 2011-2016 at a single institution. Multiparametric MRIs were acquired at 6-8 week intervals following SRS. Treated lesions were evaluated according to immunotherapy response assessment for neuro-oncology (iRANO) criteria. Tumors that were initially classified as having progression that later resolved with further follow-up and no additional intervention were ultimately classified as having pseudoprogression. Local failure was defined as progressive disease within a previous SRS treatment volume. Results: A total of 13 patients received concurrent SRS to 76 brain metastases. The median follow-up time was 11.5 months. Primary tumor types included melanoma, non-small cell lung cancer, renal cell carcinoma, and cutaneous squamous cell carcinoma. Patients were treated with SRS in 1-5 fractions to a median total dose of 22 Gy (range 19-30 Gy). ICB therapy consisted of single or dual agent therapy with ipilimumab, nivolumab, and pembrolizumab.  One patient experienced pathologically proven local failure and underwent craniotomy. There were no other local failures. Five patients (38%) experienced pseudoprogression at a median interval from SRS of 3 months (range 2-12 months). Conclusion: The majority of tumors treated with combination SRS and ICB are ultimately controlled, but pseudoprogression is a common phenomenon in these patients. The distinction between pseudoprogression and true progression has important implications for further treatment decisions and prognosis. Further research is needed to accurately differentiate pseudoprogression from true tumor progression in these patients.
Abstract Title : Bromodomain-containing protein 4 (BRD4) regulates CD8+ T cell differentiation during viral infection
Abstract : During chronic viral infections, such as HIV and hepatitis, the immune system is often incapable of eradicating the virus. CD8+ T lymphocytes play an indispensable role in the immune response against viral infections. However, during chronic infections, CD8+ T cells become functionally exhausted, characterized by an inability to kill infected cells, thus contributing to viral chronicity. Elevated expression of the inhibitory protein, programmed cell death 1 (PD1), is a prominent feature of CD8+ T cell exhaustion and drives CD8+ T cell dysfunction. Clinically, blockade of PD1 signaling restores CD8+ T cell function and improves viral clearance. Preliminary studies in our lab indicate that the chromatin modifier bromodomain-containing protein 4 (BRD4) may repress PD1 expression. We hypothesize that knockdown of BRD4 will result in the subsequent upregulation of PD1 during chronic viral infections, exacerbating CD8 T cell exhaustion and perpetuating viral persistence. The goal of this research is to investigate the role of BRD4 in controlling PD1 expression and CD8 T cell exhaustion. Understanding how PD-1 expression is regulated will ultimately yield better strategies to treat chronic viral infections.
Abstract Title : Topical Therapy of IL-10 Using a Thermosensitive Drug Delivery Platform to Treat Murine Colitis
Abstract : Background: Systemic therapies for inflammatory bowel disease such as biologics are effective, but carry increased risks of infections and malignancies. Topical therapy is effective at treating colitis while reducing systemic exposure, but can be challenging to retain. We have developed a thermosensitive delivery platform (TDP) to administer therapeutics that is liquid at room temperature, ensuring proximal delivery; at body temperature, it rapidly becomes a mucoadhesive gel, allowing for easier retention. We have shown that TDP can effectively deliver established topical therapies and now explore its use in delivering protein-based therapeutics. IL-10 holds promise as a treatment for colitis, but systemic testing has been limited by factors including the inability to reach appropriate tissue concentrations, while avoiding systemic toxicities. Here we explore TDP to deliver IL-10 (IL-10/TDP) topically to treat colitis in a murine model. Aim: To determine the ability of TDP to delivery IL-10 and the therapeutic effect of IL-10/TDP in a murine colitis model. Materials and methods: To test whether bioactive IL-10 can be released from TDP, we first measured the IL-10 concentration eluted over time in diffusate from IL-10/TDP formulation. We then tested the ability of the eluted IL-10 supernatant to suppress the pro-inflammatory response of stimulated splenocyte derived T cells. We utilized the dextran sodium sulfate (DSS) murine model of colitis to test the efficacy of rectally delivered IL-10/TDP compared to that of liquid IL-10 enema (IL-10/vehicle) and vehicle enema controls. Body weight, colon length, and histology were used to assess response to treatment. Results: IL-10 released from TDP retains bioactivity and suppressed TNF-a expression in cultured spenocytes. Results from our colitis studies show IL-10/TDP to be superior to liquid IL-10 enema (IL-10/Water) and vehicle controls in treating colitis, as demonstrated by weight recovery, colon length, and histopathology. We also tested the effect of topical IL-10 on pro-inflammatory response by assessing the intracellular cytokine profiles of CD4+ effector/memory T cells of the mesenteric lymph nodes. Intracellular levels of TNFα, IL-17, and IFNγ indicated a trend in reduction of pro-inflammatory cytokines in mice treated with IL-10/TDP compared to IL-10/vehicle and vehicle control. Conclusion: These data collectively support strong therapeutic potential for IL-10/TDP. Being a mucoadhesive gel that is easier to retain, TDP would reduce side effects/risks that can accumulate over time with systemic therapies. In addition, by overcoming retention and urgency issues with existing enema therapy, TDP can improve quality of life for people using topical therapy and potentially reduce dosing frequency.
Abstract Title : Sex differences in Toll-like receptor regulation of arthritic pain in mice
Abstract : Autoimmune diseases like rheumatoid arthritis are regulated by both adaptive and innate immune systems. We previously described the persistence of mechanical allodynia in male mice after the resolution of swelling in a passive serum transfer model of arthritis. This persistent allodynia had pharmocological features of neuropathic pain in that it was responsive to gabapentin, but not non-steroidal anti-inflammatories or anti-TNF. We found that Toll-like receptor 4 regulated the onset of this allodynia. In my project, I aimed to evaluate the role of other Toll-like receptors in arthritic pain and the differences of pain and swelling between male and female mice. Using the K/BxN serum transfer model of murine arthritis, I tested a series of mice that were deficient in single or multiple Toll-like receptors and their signaling pathways. Male and female mice were tested and compared. Wild-type female mice can resolve their pain. However, other strains of Toll-like deficient mice cannot resolve their pain in the post-inflammatory phase.
Abstract Title : P2XR and Caspase Deficiency Affect Arthritis in Males more than Females
Abstract : Rheumatoid arthritis (RA), characterized by joint inflammation and destruction, is a chronic and systemic autoimmune disorder. One promising therapeutic target was ATP-gated P2X7 Receptor Cation Channel (P2X7). Extracellular ATP signaling acts through the P2X7 receptor and induces the release of proinflammatory factors (e.g., interleukin-1β, prostaglandins, and proteases) associated with the clinical symptoms of RA. Despite early promise oral antagonists were not effective in a clinical trial. Using the K/BxN serum transfer model we tested P2XR deficient male and female mice that demonstrated similar levels of paw swelling to their wild type counterparts. Caspase 1 is a critical enzyme for IL-1 activation after P2XR stimulation. These knockout mice also displayed swelling similar to wild type mice. However in double deficient mice there was a reduction in paw swelling, which was greater in males than females. Although P2XR antagonism and IL-1R decoys are not generally effective as monotherapies in RA and a combination strategy may be more effective. In addition gender maybe a factor in therapeutic decision-making.
Abstract Title : The Role of Gene PLD3 in the Immune System
Abstract : Enzymes within the phospholipase D (PLD) family have traditionally been known to catalyze the hydrolysis of phosphatidylcholine, a key component of the plasma membrane of mammalian cells. However, they may also possess other functions. Our research aims to uncover these functions, particularly of isoforms PLD3 and PLD4. I specifically studied PLD3 and employed the CRISPR-CAS9 system to create PLD3 mouse knockouts to help identify changes in the immune system. PLD3 is highly expressed in brain tissue, and genome-wide association studies have linked PLD3 variants to Alzheimer’s disease. My results confirm that my CRISPR constructs successfully knocked out mPLD3 in-vivo. Specifically, I observed a base insertion that resulted in a reading frame shift. This would effectively eliminate PLD3 protein function by introducing a premature stop codon. ELISA testing also suggests that PLD3 and 4 affect IFN-1 response, which could contribute to neuroinflammation. In addition, PCR splicing by overlap extension (SOEing) was used to mutagenize basic residues of mPLD4 protein, as these might be facilitating binding of anionic substrates.