Tuberculosis is a major infectious disease with approximately 2 billion people infected worldwide. With the emergence of multi-drug resistant tuberculosis (MDR-Tb), efforts to develop efficient treatments with fewer cytotoxic effects are currently under investigation. Dual host and pathogen directed therapeutics hold promise towards more effective treatments against tuberculosis (Tb) through mechanisms that enhance bacterial clearance and reduce tissue damage and immunopathology. We investigate RGFP966, a drug that modulates the host's immune defenses and directly reduces Mtb growth (a histone deacetylase 3 (HDAC3) inhibitor). The aim of my project is to characterize the effect of RGFP966 on Mtb growth. My hypothesis is that the antimicrobial effects of RGFP966 are specific to Mycobacterial species, and the bacterial target of RGFP966 is required for its survival. Within the experiential growth period, I treated Mycobacterium avium and M. Smegmatis in 7H9 broth with a range of RGFP966 concentrations (0.5 uM, 5 uM, and 50 uM) and incubated in culture tubes and 96-well plates for 3-7 days. Within designated time intervals, I analyzed levels of dose dependent inhibition using optical density, luminometry, and fluorometry methods. Current results through optical density and luminometry demonstrate that RGFP966 controls M. Avium growth in 7H9 broth with an MIC50 of approximately 5 uM. Fluorometry and optical density results indivate no restriction of M. smegmatis growth. Comparing these results to previous findings in our laboratory, where RGFP966 lacked effects against common gram-positive and gram-negative bacteria, indicates possible specificity of the antimicrobial effects of RGFP966 to Mycobacterial species. We will further examine the effects of RGFP966 against mycobacterial growth. A comprehensie understanding of its pathogen directed effects combined with its host directed effects hold promise as an adjunctive therapeutic against Tb infection. 

We previously observed that turmeric could be used to manipulate gut motility in a microbiome-dependent-bile acid-mediated manner via the enteric nervous system (ENS). Bile acids comprise a class of metabolites synthesized by the host and modified by gut microbes. Bacterial bile salt hydrolases (BSH) are responsible for deconjugation, the first step in bile acid metabolism for generation of secondary bile acids. We hypothesized that homeostatic ENS signaling is dependent upon gut bacterial bile acid metabolism. To test this hypothesis, we colonized wild-type gnotobiotic mice with different defined consortia varying in BSH activity and subjected them to a two-week low-fat diet ± turmeric. As a control, one treatment group remained germ-free. mRNA isolated from small intestine and colon was subjected to gene expression profiling returning counts of 68 target genes, including ENS-specific genes, and 7 housekeeping controls. A machine learning algorithm was deployed to identify genes whose expressions were most impactful in discriminating between treatment groups. Glp2r and VIP were highly discriminatory with respect to BSH activity. Enteric neurons express Glp2r, activate the mTORC1 pathway in response to GLP-2, and modulate intestinal epithelial cell growth. We found that Glp2r expression was significantly greater in mice harboring consortia with BSH activity than in germ-free mice. GLP-2 induces VIP neurotransmission from enteric neurons. Indeed, VIP expression is correlated with Glp2r expression. These data suggest that gut bacterial bile acid metabolism regulates homeostatic ENS signaling, with implications for gut motility and colorectal carcinogenesis.

African Americans may be at increased risk of developing Alzheimer's disease. Decreased social engagement and depression are related; changes in older adult levels of social engagement are associated with dementia. The Sharing History through Active Reminiscence and Photo-Imagery (SHARP) study aims to improve cognitive health among cognitively normal and mildly cognitively impaired older African Americans by employing a culturally celebratory framework integrating walking and community-based social reminiscence. In triads, African Americans aged >55 (n=40) walked 3x/week for 6-months in 2016 and 2017 while engaging in image-prompted conversational reminiscence about neighborhood history. Walks took place in Portland, Oregon’s historically Black neighborhoods. Baseline and end-study surveys had seventeen close-ended questions about physical and mental health and health behaviors. SPSS analytical software will aid in analysis. Focus groups discussed the impact of the program’s culturally celebratory approach on physical activity and mental health. Qualitative data will be thematically coded. Preliminary data highlights self-reported health-related outcomes which include increased activity throughout the day on days that walks occurred, rekindled friendships, increased impromptu social engagement, and heightened sense of community connectedness as a result of increased social engagement and place-based group reminiscence. Participants feel that the program’s social aspect, rather than physical activity, is the primary driver for sustained engagement. Structured socializing and the focus on neighborhood history contributes to improved self-reported mental health, important for sustained cognitive health. Finding innovative, culturally meaningful ways for older adults to be socially engaged may improve mental and cognitive health outcomes.

 Esophageal adenocarcinoma (EAC) has received considerable attention recently due to a three-fold increase in occurrence affecting 20,000 people each year in the US with <20% patients surviving 5 years past diagnosis. Thus, there is an urgent need to advance our understanding of EAC’s molecular pathology and to use this understanding in improving our ability to prevent and manage EAC. Gene mutations and epigenetic alterations drive the formation of a pre-malignant condition called Barretts’ esophagus (BE), which can then progress to EAC. Epigenetic modifications of numerous genes, in the form of DNA hypermethylation with subsequent gene silencing, have been demonstrated to occur frequently in BE and have been postulated as a driving force for EAC formation. In prior studies, we discovered a novel methylated gene, TUSC1, with silenced expression in primary EAC tissue samples and cancer cell lines (Yu et al., Gut 2018). This study aims to investigate the biological consequence of TUSC1 loss in EAC cell lines by utilizing CRISPR-Cas9 gene editing technology. We hypothesize that TUSC1 is a novel tumor suppressor gene in EAC. To test this hypothesis, we will be generating TUSC1 knock-out (KO) EAC cells by introducing TUSC1-silencing guide-RNA into EAC cell culture. We will be assessing the extent of TUSC1 KO at the gene expression and protein level by conducting quantitative real-time PCR and Western blotting, comparing the KO cells to the control cells. We will also use PCR-based sequencing to verify the DNA alteration introduced by CRISPR in single-cell colonies. Successful completion of these experiments will lead to the next stage of phenotypic assays, in which we will investigate whether loss of TUSC1 in EAC cells would promote oncogenic behaviors of cancer cells. The proposed studies would demonstrate the biological function of TUSC1 in EAC and illustrate the molecular mechanisms of DNA-methylation driving EAC tumorigenesis.

Alzheimer’s disease (AD) is a neurodegenerative illness affecting millions of individuals in the United States and represents the leading cause of dementia worldwide. AD is pathologically defined by abnormal accumulation of interneuronal plaques composed of amyloid beta protein and intraneuronal tangles composed of tau protein. The focus of our current research involves understanding how tau protein homeostasis is restored using the model organism C. elegans. One process by which protein homeostasis is modulated is through the endoplasmic reticulum unfolded protein response (UPRER), which is composed of three signaling branches. Initially, the UPRER acts to restore normal protein folding, but if restoration is impossible, it triggers apoptosis. Our lab became interested in this signaling pathway after we identified the UPRER master transcription factor XBP-1s as a modifier of tau pathology (tauopathy). Specifically, XBP-1s overexpression protects against tauopathy in our C. elegans model. Previous RNA sequencing (RNAseq) analysis identified several downstream target genes upregulated with XBP-1s overexpression in our pathological tau C. elegans model. To follow up, I am working on understanding the molecular basis of XBP-1s-mediated tauopathy suppression by generating C. elegans models containing target genes of interest. Assessment of the effect on tauopathy phenotypes includes behavioral analysis by performing a liquid thrashing assay and tau protein analysis via the Western blot technique. Using the microinjection technique, I have generated a transgenic C. elegans model overexpressing hsp-4, an upregulated XBP-1s target gene identified by RNAseq that potentially modulates tauopathy. We are currently determining whether overexpression of hsp-4 can ameliorate tauopathy similar to XBP-1s overexpression. Gaining further information about the role of these XBP-1s target genes in tauopathy using the model organism C. elegans is valuable for future translational studies in mammals, along with being integral to research for neuroprotective therapies for neurodegenerative diseases such as AD.

Merkel cell carcinoma (MCC) is a rare and aggressive cutaneous malignancy with a high propensity for recurrence and distant metastasis. Individuals with chronic immunosuppression have both a higher predilection towards developing MCC and tend to have a more aggressive disease course. Chronic lymphocytic leukemia (CLL) is among the most common types of immunosuppression associated with MCC. Immune-checkpoint inhibitors (such as anti-PD1 or anti PD-L1 therapies recently approved for cancer treatment) are associated with improved disease-specific survival and are often used to treat patients with progressive, metastatic MCC. However, the effectiveness and side-effect profile associated with treating metastatic MCC in CLL patients with immunotherapy is not well categorized. This study seeks to understand the risk-benefit profile of immunotherapy in this setting, and a possible combined role for radiation in the treatment of CLL patients. Data was abstracted from a Seattle-based prospective registry of 1,439 MCC patients in which 9 patients were identified to have had CLL and been treated with immunotherapy. Patients were assessed for side effects of immunotherapy, progression of disease and survival status. Six patients had side effects from immunotherapy with 5 of the patients having side effects that resulted in termination of treatment. The average number of doses of immunotherapy received before termination was 3. Eight patients had progressive disease after the initiation of immunotherapy, 4 of whom have died of progressive MCC. No patients have had a complete and ongoing response to immunotherapy. These findings suggest that despite the efficacy of immunotherapy in immune-competent MCC patients, this approach may be less effective in CLL patients than immunocompetent patients. In additional, the side effect profile seemed to be more frequent in CLL-MCC patients. Neutron radiation and combination therapy are discussed as potential treatment options. Further investigation into treatment options for MCC patients with immunosuppression, such as CLL, is needed.

In 2009, the Department of Veterans Affairs (VA) integrated Store-and-Forward Teledermatology (SFT) to increase the dermatology care to patients with limited access. With this, patients are evaluated by their primary care provider (PCP) and referred to SFT for dermatology conditions. A technician on site takes images of the condition and send records to tele-dermatologist at a reading hub to evaluate and provide a differential diagnosis, treatment, and follow up. When skin biopsies are requested, Clinical Pathological Correlation (CPC) consultations are also requested. Dermatopathology reports are not standardized well and often require interpretation in context of other clinical information, including history, imaging, and microbiology studies to diagnose. Follow up imaging after an interval of treatment may be requested to confirm the diagnosis. The purpose of this quality improvement study is to assess adherence of PCPs to follow ups, determine whether failure to use means degraded clinical outcomes and interventions to improve outcomes. We conducted a retrospective review of 224 conditions addressed by teledermatology during the month of April 2018, on Veterans in the Pacific Northwest Network (VISN 20) where recommendations for either CPCs, re-imaging, or both were requested. We searched electronic medical records that requested follow-ups and determined a.) whether it occurred (if not, why); b.) timing it occurred; c.) affectability of treatment plans based on follow up findings; and d.) whether conditions were by intervention. Of 224 conditions reviewed, 161 requested CPC consultation, 63 requested reimaging, and 2 requested both. For CPC consultations, 90.06% patients had a re-consult, through CPC process or FTF and 9.4% of cases were lost in care. For reimaging consults, 73% of cases had reimaging or FTF re-consult while 27% of cases were lost in care. Data from this project will be used to improve follow up and quality of care Veterans receive through SFT.

Merkel cell carcinoma (MCC) is a rare and aggressive skin cancer with a recurrence rate of ~40%. The Merkel cell polyomavirus (MCPyV) is causally linked to 80% of MCC cases, while the remaining 20% are caused by UV-induced mutations. A blood test has been developed to detect antibodies to the MCPyV oncoprotein, as these antibody levels have been shown to correlate with disease burden. This test is a useful tool for tracking disease recurrence in patients who produce antibodies and is recognized by the 2018 National Comprehensive Cancer Network (NCCN) Guidelines for this purpose. However, as systemic immunotherapies are increasingly integrated into the standard of care for patients who develop metastatic disease, it is critical to understand the impact of immuno-stimulatory drugs on oncoprotein antibody levels. Using a Seattle-based MCC repository of 1,444 patients, I identified 64 patients who produced antibodies to the MCPyV oncoprotein and received immuno-therapeutic treatment. Eighteen of these patients had serial antibody tests during treatment, providing sufficient data for analysis. To establish a comparison for measuring disease status, I identified imaging studies from patient medical charts, such as PET/CT and MRI, or clinical evaluations administered within 45 days of the antibody test. The imaging studies were used as comparison due to their efficacy and reliability in disease detection. Among all 18 patients, those with increasing tumor burden, had increased antibody titers, while those with decreasing tumor burden had falling or low antibody titers. These results suggest that this antibody test is also an effective indicator of recurrence in MCC patients who are receiving immunotherapy.

Amyotrophic Lateral Sclerosis (ALS) is a neurodegenerative disease that results in progressive degeneration of upper and lower motor neurons, leading to muscle weakness, paralysis and death. Currently, ALS affects 15,000 Americans in the U.S., who have an average life expectancy of three to five years at the time of diagnosis. There are only two FDA-approved medications for ALS treatment, which minimally slow but do not reverse the progression of the disease. Understanding the genetic causes of ALS can help us to identify more effective treatments, therefore we seek to identify pathogenic variants and investigate how they contribute to the death of motor neurons. We previously showed that a tandem repeat in an intron of the gene WD-repeat-containing protein 7 (WDR7) may be involved in ALS. To better characterize this repeat, we PCR-amplified the repeat region and quantified repeat size in 500 ALS samples, including those from sporadic cases and those with known pathogenic variants. These repeat sizes were compared to approximately 500 Parkinson disease samples, 100 Primary Lateral Sclerosis samples, and 500 control samples from the Coriell Cell Repository, to verify whether expansion of this repeat was specific to ALS. Furthermore, to determine if repeat blocks were enhanced in certain subsets of patients, the lengths of the intronic repeats in genomes of ALS patients were profiled and compared with de-identified phenotypic information. To resolve the exact sequence of repeat in ALS samples, a subset of patient DNA samples and controls were sequenced using single molecule real-time (SMRT) sequencing. Together, these findings help us gain insight into the disease and guide us to develop a better treatment.

Ovarian cancer is the deadliest gynecological malignancy: nearly 70% of patients are diagnosed at late stage and 70% relapse. Cytoreductive surgery and chemotherapy are the standard of care, yet fewer than 50% of patients live 5 years past diagnosis. Innovative therapies are desperately needed. Immunotherapy using T cells engineered to express a T cell receptor (TCR) targeting proteins overexpressed in tumors is a promising strategy that limits tumor growth and has minimal toxicity. Mesothelin (Msln) is overexpressed in over 75% of ovarian cancer patients and has been identified as a promising cancer antigen because it contributes to the malignancy and invasiveness. Our group has shown that targeting Msln with engineered T cells (TCRMsln) in the mouse ID8 ovarian tumor model, which recapitulates essential aspects of the human disease, can prolong survival of mice with advanced disease. However, our data also show that immunosuppressive features in the tumor cause T cells to become dysfunctional and/or die. Fas ligand (FasL) is a transmembrane death-inducing protein present in ovarian tumors, and may be used by tumor cells to escape the immune response by inducing cell death of beneficial lymphocytes expressing the Fas receptor (Fas). We hypothesized that we could overcome this T cell evasion mechanism using immunomodulatory fusion proteins (IFP). We generated IFPs that combine the Fas extracellular binding domain to a co-stimulatory domain, CD28 or 4-1BB, instead of the standard death domain, to trigger T cell activation. T cells expressing an IFP had enhanced proliferation and persistence in ID8 tumors relative T cells lacking an IFP. Further, ID8 mice treated with TCRMsln T cells expressing an IFP had prolonged survival compared to mice treated with the TCRMsln T cells alone. Many solid tumors overexpress FasL, thus our findings have the potential to enhance engineered T cell therapy against other malignancies.

 Many veterans live in rural areas with limited access to traditional face-to-face (FTF) dermatological services. This barrier to care can result in worse health outcomes. Store-and-Forward Teledermatology (SFT) is a program that aims to address this disparity by providing veterans with increased access to dermatological care. Instead of seeing a dermatologist in person, veterans with suspicious lesions first see a primary care physician (PCP) who then arranges for pictures of the lesions to be taken. These pictures are electronically sent to a dermatologist who reviews them remotely and then suggests a plan of care for the patient. SFT allows veterans to both avoid potentially long wait times at in-person dermatology offices and address lesions that may have gone untreated. This study focused on the timeline of care for the treatment of melanoma. Because of its lethality, it is important to diagnose and treat melanoma as quickly as possible. In this study we compared the timeline to treatment – from when the lesion was first discovered to the date of its surgical excision – in groups receiving standard FTF dermatological care and SFT care. Using chart data from patients within the VISN 20 network, we retrospectively gathered the dates of discovery, biopsy, and excision in patients. From there, we calculated the mean days from discovery to biopsy and biopsy to excision in both groups. Upon comparing these timelines to each other we found that the overall timeline for SFT was approximately 12 days longer than that for FTF. We identified sections of the SFT process that may be responsible for the delay as potential quality improvement points in the future.

Cardiovascular disease (CVD) continues as the leading cause of death worldwide. The underlying cause of CVD is atherosclerosis, characterized by fatty plaques in the inner walls of the artery, and exacerbated by oxidative stress, inflammation and immune cells. The high-density lipoprotein (HDL)-associated enzyme paraoxonase-1 (PON1) plays a significant role in protecting against CVD. However, the mechanism of PON1’s protection is not well understood. Our laboratory and others have reported a progressive decrease of PON1 activity in several diseases. Our goal is to understand how PON1 prevents oxidative stress and its potential measurement as an early biomarker of risk of disease. Our hypothesis is that oxidative stress inhibits PON1 enzymatic activity, leading to progression of the atherosclerotic process in CVD. The objective of this research is to generate a mouse model to allow us to study the human enzyme PON1 in induced atherosclerosis in vivo.  Starting from two strains of knockout (KO) mice, we have generated the Pon1/apolipoprotein E (apoE) double KO mouse, known to be susceptible to atherosclerosis. We are now crossing this double KO mouse with Pon1 KO mice that express human PON1 (transgenic human PON1 mice, tgHuPON1). We collect ear punches for DNA extraction and blood from the saphenous vein for activity assays. We use polymerase chain reaction (PCR) genotyping methodology, which amplifies DNA, to identify mice that are KO for mouse Pon1 and apoE and that express human PON1. We run enzymatic assays to determine activity levels of the human PON1 in mice. Once we have created the tgHuPON1/apoE KO mouse, we will feed mice with an atherogenic diet, which will induce subclinical atherosclerosis. This knowledge will contribute to understanding the relationship between PON1 and CVD, and will likely generate a useful biomarker for risk of disease.      

Polycystic kidney disease (PKD), commonly caused by defects in polycystin-1 or polycystin-2, results in the formation of fluid-filled cysts and progressive loss of kidney function. Our laboratory has established a cellular model for ADPKD using kidney organoids, multicellular tissue that functionally and structurally resembles the organ of interest, derived from gene-edited human pluripotent stem cells (hPSC). To address the need surrounding a cure for PKD, we have also discovered a basis for a form of treatment involving myosin. Non-muscle myosin is a protein that controls cell protrusion and adhesion; we have established that lack of adherence increases cystogenesis dramatically. We discovered blebbistatin, a non-muscle myosin (NMMII) inhibitor, is a robust inducer of cystogenesis. Our previous finding begged the question if activators of NMMII have the opposite effect and reduce cystogenesis in PKD organoids. To better understand the role of myosin, we differentiated kidney organoids from human PKD hPSCs in a 24-well plate coated with Matrigel. We proceeded with microdissecting the organoids at 3 weeks before they formed any cysts and transferred the organoids to suspension culture where they were treated with either DMSO as a control or a pharmacological myosin activator for one week. The organoids were then imaged and number of cystic organoids were quantified. The treatment using the myosin activator compound resulted in decreased cystogenesis, in terms of the size and number of cysts. Although we have yet to clarify myosin’s effect in the PKD pathway, we have identified a myosin activator as a potential inhibitor of cystogenesis. Further experimentation is to be done with other compounds such as actin activators, and proceeding with experimentation on ex vivo kidneys of mice. Our current findings suggest that the polycystin proteins positively regulate actomyosin’s contractility, therefore myosin may be an important factor for keeping kidney tubule integrity and preventing cystogenesis.

Treatments for cystic fibrosis (CF) have extended patients’ lifespan, resulting in CF-related diabetes (CFRD) as a major CF complication affecting 30-50% of adults. A key pathological feature of CFRD is the deposition of islet amyloid polypeptide (IAPP) as amyloid in pancreatic islets. In type 2 diabetes, Islet amyloid is associated with decreased beta-cell mass and function. Current mouse models of CFRD do not develop all the pathological features observed in human CFRD, including islet amyloid. To generate a mouse model of CFRD which exhibits islet amyloid deposition, we crossbred a CF mouse (CftrF508del) and a human IAPP (hIAPP) transgenic mouse and compared amyloid deposition in cultured islets from the resulting offspring. Islets from each of the mice genotypes (NT.Cftrdel/del (n=2), NT.Cftr+/del (n=4), NT.Cftr+/+ (n=5), hIAPP.Cftrdel/del (n=2), hIAPP.Cftr+/del (n=5), hIAPP.Cftr+/+ (n=3)) were isolated and cultured for 7 days in high (16.7 mM) glucose to induce amyloid formation. Islets were fixed in neutral-buffered formalin, paraffin-embedded and sectioned. Sections were stained with thioflavin S (for amyloid) after which amyloid prevalence (% islets with amyloid), amyloid severity (% islet area occupied by amyloid) were quantified. Islets from hIAPP transgenic mice developed islet amyloid when cultured in vitro, while islets from non-transgenic mice did not. Among hIAPP transgenic mice with different CF genotypes (hIAPP.Cftrdel/del, hIAPP.Cftr+/del, hIAPP.Cftr+/+, respectively), islet amyloid prevalence (74±13%, 73±11%, 81±5 %; p=0.89 by ANOVA)[NE2] or severity (3.4 ± 1.7%, 2.0 ± 1.1%, 3.57 ± 0.4%; p=0.64 by ANOVA) did not differ. The presence of amyloid in islets of mice expressing hIAPP genotypes supports the utility of this new mouse model to study this aspect of islet pathology as seen in human CFRD.

Polycystic kidney disease (PKD) affects nearly 600,000 people in the United States and it is caused by mutations in the genes that encode polycystin-1 (PC1) and polycystin-2 (PC2). These modifications are responsible for the growth of fluid-filled renal cysts that can impair kidney function over time, and lead to end stage renal disease. PC1 and PC2 have also been localized to the mitochondria, suggesting that they are responsible for regulating cellular metabolism and energy production, which can lead to mitochondrial dysfunction and increased PKD progression. Our lab uses human pluripotent stem cells (hPSCs) to generate kidney organoids that can be used to study the advancement of PKD, which contain the major proximal structures of the nephron including podocytes, proximal tubules, distal tubules, and endothelial cells. Orgnaoids that lack both PC1 and PC2 will develop cysts that can grow large enough to be seen by eye. Unfortunately, these organoids lack a collecting duct (CD) system, a crucial component of the kidney that expands greatly in PKD. To address this need, we developed a protocol to differentiate hPSCs into ureteric bud (UB) cells, the precursors of CD cells. First, we identified Dolichos biflorus agglutinin (DBA) as a key CD lineage marker in developing kidneys through immunofluorescence analysis on human kidney tissue. Then, we took undifferentiated hPSCs and investigated their ability to express DBA by treating them with a concentration gradient of small molecules known to induce kidney lineage cells. The resulting cells were stained with DBA and GATA 3, an additional, early CD marker, and we found that both markers were expressed in vitro. We will continue modifying this protocol to better study the increased metabolic rates seen in PKD, its effects in CD cells, and the mechanism behind the PC1 and PC2 pathway that leads to cyst formation.

In the central nervous system, insulin acts as an anorexigenic hormone, regulating the desire for fatty and sugary foods. It also plays an important role in learning and memory. Thus, a malfunctioning insulin transport system across the blood-brain barrier (BBB) could be linked to obesity, the development of type 2 diabetes, and cognitive impairments as occur in Alzheimer’s disease. Studies have shown that insulin binds to insulin receptors (IR) located on the endothelial cells which make up the BBB; the activated insulin-IR complex is then taken up into the cell, where it sets off a signal cascade. Additionally, insulin binds a protein responsible for transport from the luminal to abluminal side of the brain endothelial cell. However, the mechanism by which this occurs is still not understood. Thus, we set out to elucidate the difference between insulin receptor uptake and insulin transcytosis in vivo, focusing on clathrin and caveolin, the two proteins primarily responsible for mediating endocytosis. To do this, we use radiolabeled insulin, the IR antagonist S961 (which binds to IR but is not taken up into the cell), and three pharmacological agents: phorbol 12-myristate 13-actetate (PMA) to promote endocytosis, monensin to disrupt clathrin-mediated endocytosis, and filipin to disrupt caveolin. To eliminate serum factors, cardiac perfusion in male CD-1 mice will be performed. One group will be the vehicle control group, and the other will receive radiolabeled insulin, S961, in addition to one of the three pharmacological agents. Perfusions will last from 1-10 minutes, in which brains are collected and dissected into brain regions. Radioactivity is measured in the hypothalamus and hippocampus as well as whole brain. The data collected will help us better understand the differences between insulin transcytosis versus endocytosis and how insulin transport may go awry in Alzheimer’s disease, diabetes, and obesity conditions.

A patient’s performance status (PS) is a key determinant of eligibility for enrollment in acute myeloid leukemia (AML) clinical trials, as PS is regarded as a crucial predictor of death as a result of treatment on the trial. PS values range from 0 (minimally symptomatic) to 4 (bed-ridden). It is crucial to account for possible differences in PS when comparing results of different trials. However, the degree of concordance between observers in assessing PS remains unknown. Because of this uncertainty and the clinical significance of PS, my research studies inter-observer concordance in assessing PS. Specifically, I compare PS as assigned in electronic medical records by medical providers, as abstracted by myself given history of present illness and physical exam findings, and the patient’s own evaluation via a health questionnaire. Data was collected from 300 randomly selected patients arriving to Seattle Cancer Care Alliance from January 2015 to December 2017 with newly diagnosed AML. The results of this study may inform similar research within other treatment centers, and development of increasingly precise guidelines for evaluation of PS to improve reliability.

A positron emission tomography scan (PET scan) can be used to map areas of potential cancer in the body. Cancer cells multiply at an incredibly fast rate, and in doing so, use a greater amount of glucose to fuel their high-energy needs compared to normal tissues. PET scans use radiolabeled fluorodeoxyglucose to quantify cellular metabolic activity, producing visual hotspots that are usually overlayed on computed tomography (CT) images and correlate to cancer sites (“PET/CT”). PET/CTs are often used to follow patients with acute myeloid leukemia (AML) after hematopoetic cell transplantation (HCT). In the lab’s database of over 300 consecutive patients whose AML relapsed after HCT, we found 15 patients who were followed with PET/CT because of extramedullary disease. These patients received a variety of treatments for their AML relapses, including induction chemotherapy, radiation, and/or hypomethylating therapy. We sought to assess if a negative post-treatment PET was helpful in predicting a cure. Through detailed chart review, we found PET scan negativitity is hard to achieve, a negative PET is not reassuring (50% still relapsed) and even a low SUV-positive PET is prognostic of disease progression. Moreover, if any extramedullary disease is present, systemic therapy prolongs life more than local radiation alone, but no treatments are likely to be curative.

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by multi-organ inflammation and damage, including skin and kidney. Ultraviolet B light (UVBL) is the only environmental factor known to precipitate both skin and kidney disease. How UVBL-triggered sterile inflammation in the skin influences kidney injury remains a pressing question for SLE patients, about 70% of whom suffer from sensitivity to UVBL. We previously demonstrated that neutrophils are the first immune cells to infiltrate the inflamed skin tissue after exposure to UVBL. To investigate the neutrophils’ role in UVBL-induced kidney injury, C57BL/6J mice were irradiated with a single dose of UVBL (500mJ/cm2). Cells in the bone marrow (BM), skin, blood, and kidney were characterized using flow cytometry. Gene expression of inflammatory mediators and adhesion molecules was evaluated using qPCR. Following acute exposure to UVBL, we observed a 10-fold increase in skin neutrophils, associated with a decline in neutrophils from the BM and a 5-fold increase in circulating neutrophils relative to baseline (no UVL). Relevant to SLE, neutrophils increased up to 10-fold in the kidney after skin UVBL injury (vs. no increase in monocytes/macrophages). Local skin response was characterized by rapid induction in inflammatory cytokines (IL1b, TNFa, IL6, IL33) and neutrophil chemoattractants (G-CSF, CXCL1, LIX) (day 1-2) that returned to baseline by day 6 after UV. Neutrophil infiltration into the kidney was accompanied by endothelial activation and inflammation: increased VCAM1, E-Selectin, IL1b, Ngal, and s1008/9 gene expression, markers of kidney injury in SLE, as well as by transient proteinuria. Neutrophils recruited to the kidney demonstrated two phenotypes: early activated CXCR2hi (day 1-2) and late aged CXCR4hi (day 2-6), which followed expression of CXCR4 ligand CXCL12, another marker of kidney injury. Together, our findings propose a novel neutrophil-dominated skin-kidney axis of pathogenesis and provide a model for UVBL-triggered disease UW flares in SLE.

African-born individuals in the United States face unique barriers in accessing preventative healthcare. We conducted a mixed-methods study to implement community-based health fairs and learn in the process the barriers regarding accessing preventative healthcare. From April to May 2018, we held six health fairs at three apartment complexes with high numbers of African-born residents. Fairs included free point-of-care screening for glucose, cholesterol, BMI, blood pressure, and HIV along with social services and health education. The health fairs were hosted in apartment complex common areas with HIV testing conducted in private rooms. Health fair participants were asked to complete a series of questionnaires to evaluate demographics, access to health services, and HIV testing history. We conducted 18 key informant interviews with health fair participants and community leaders to identify barriers to preventative healthcare among African-born individuals. Of the 111 adults who accessed at least one service at a health fair, 92 completed questionnaires. 61% were female, 52% were born in Africa, and 63% had health insurance. Among the 18 interviewed, the most commonly reported barriers were language, logistical barriers, a distrust of health professionals, and lack of health literacy. These barriers create a disconnect between patients and health providers and impedes access to health-related information. A high prevalence of non-communicable diseases was also identified among participants, including obesity, high blood pressure, and high cholesterol. Residential health fairs are a feasible method to increase engagement of African-born individuals in preventative care. Key considerations are providing culturally-competent care, easily accessible services, and staffing services with healthcare providers who speak the primary languages of the populations being served or have adequate interpretation services. Preventative healthcare services will also be more acceptable if all services (e.g. cardiometabolic and HIV screenings) are incorporated into one package and promoted as routine activities.

High-density lipoprotein (HDL) plays an important protective role in development of atherosclerosis, one of the leading causes of cardiovascular disease (CVD) and death. Although HDL is generally believed to exert anti-inflammatory effects in cells, recent data have emerged showing that HDL can enhance the pro-inflammatory effects of inflammatory stimuli under some conditions. However, molecular mechanisms involved in the pro-inflammatory effects of HDL are not well understood. We hypothesized that HDL-mediated cholesterol depletion in macrophages drives the pro-inflammatory effect of HDL in an ADAM metallopeptidase domain 17 (ADAM17)-dependent manner. Bone marrow-derived macrophages (BMDMs) were collected from wild-type mice and from mice deficient in ADAM17 in hematopoietic cells. ADAM17 is a membrane-bound sheddase that cleaves extracellular parts of several membrane proteins. The BMDMs were pretreated with HDL for 18 hours before stimulation with lipopolysaccharide (LPS). At the end of the LPS treatment (10 ng/ml, 6 hours), BMDMs were collected and used for gene expression or protein analysis. We observed that HDL (100 μg/ml) exaggerated the response of LPS on tumor necrosis factor alpha (4-fold over LPS alone) and interleukin 1 beta (2.8-fold over LPS alone) gene expression in BMDMs. This effect was prevented by cholesterol loading of the macrophages. The pro-inflammatory effects of HDL were associated with increases ADAM17 gene expression (50% over LPS alone) and protein (~2.5-fold over LPS alone) levels. Moreover, deletion of ADAM17 in macrophages prevented the pro-inflammatory effects of HDL by inhibiting HDL-mediated cholesterol depletion. We also observed that BMDMs from ADAM17-deficient mice exhibit elevated expression of genes related to fatty acid and cholesterol synthesis. Overall, these data suggest that deficiency of ADAM17 alleviates the pro-inflammatory effects of HDL by preventing cholesterol depletion in macrophages. This research helps us to understand HDL’s functions in inflammatory cells, which become dysfunctional in several pathophysiological states, including diabetes and cardiovascular disease.

Von Willebrand Disease (VWD) is a bleeding disorder in which von Willebrand Factor (VWF), a polymeric blood protein, is either completely absent or in a dysfunctional state within the circulatory system. VWF’s function is to respond to shear force through an unfolding conformational change, and it is this unfolding and consequential exposure of platelet binding sites that initiates the blood clotting cascade. Affecting 1% of the entire population, VWD is the most common inherited bleeding disorder. However, few effective treatments exist. A major barrier to understanding VWF function is the absence of human cellular and vascular models that can accurately reconstitute complex phenotypes and molecular mechanisms. Studies of VWF at the cellular and vascular scale can provide important insight into physiological factors that regulate VWF. Human cells are particularly attractive and provide a highly accessible, species-specific model that can be more flexible than mouse models. Current cellular models for VWF functional studies are primarily limited to endogenous VWF secreted from human umbilical vein endothelial cells (HUVECs), which are not immortal, making it difficult to engineer disease models. This project serves to directly address these barriers through the creation and characterization of stable VWF knock-out human pluripotent stem cell lines (hPSC). These cells are immortal and can differentiate into numerous lineages including endothelia. VWF knock-out cells differentiated normally into endothelial cells, as expected, based on cell morphology and endothelial marker expression. The VWF knock-out cells have been confirmed through immunoblot and immunofluorescence to be deficient in various VWF-associated proteins, such as Factor VIII and Angiopoietin-2. We are currently investigating the mechanisms behind these deficiencies and how they relate to the absence of VWF. These findings will enable us to better understand the function of VWF, which will ultimately guide us to discover effective treatments for VWD.

Endothelial cells within the pancreatic islet produce key factors for survival and functionality of insulin-secreting β cells. Laminin is one such factor, thought to be responsible for enhancing insulin release. Laminin is a heterotrimeric protein composed of three isoforms: α-chain, β-chain, and γ-chain. Islet endothelial cells from diabetic (db/db) mice show a significant decrease in the expression of laminin α4, β1, and γ1, and show a reduced ability to secrete insulin. To test the correlation between these in vivo observations, we used an immortalized islet endothelial cell-line (MS-1 cells), to decrease genes of interest; and generate conditioned media (CM) in which to culture isolated islets to test their insulin secretion. Islets cultured in CM from MS-1 cells with decreased laminin α4 expression did not have blunted insulin release. However, we found that that decreased laminin α4 increased expression of laminin α1, perhaps as a compensatory mechanism. We inhibited expression of laminin α4 and α1 in MS-1 cells, and cultured islets exposed to this CM also did not have decreased insulin secretion. Therefore, we hypothesize that reducing all three isoforms of laminin in MS-1 cells is necessary to cause a decrease in insulin secretion in islets exposed to this CM. To determine if the knockdown of laminin-411 is sufficient to impair islet insulin release, CM will be collected from MS-1 cells exposed to oligonucleotides designed to inhibit the expression of laminin-411 or a nonspecific control oligonucleotide for 24hrs. After the media is collected, glucose concentrations of the two types of CM are matched. Isolated islets are exposed to these CMs for 48hrs and insulin secretion will be determined for basal and glucose stimulated conditions. We anticipate that CM generated by cells with reduced laminin 411 to decrease insulin secretion in isolated islets, replicating the results observed from db/db mice.

Utilizing the Type III Secretion System (T3SS), Shigella spp. uses a cascade of proteins to manipulate, penetrate, and colonize host eukaryotic cells. Inducing epithelial necrosis, Shigella spp. infection is responsible for moderate to severe diarrhea in millions of children and immunocompromised individuals — the majority from low and middle income countries. Previous translational research for Shigella spp. has been limited due to a lack of robust in vitro and in vivo models to screen therapeutic efficacy. Understanding infectivity of Shigella spp. heavily relies on imprecise estimations of intracellular Shigella spp., which ultimately impacts vaccine and antibiotic efforts. This project aims to address this problem by developing a novel red intracellular reporter to quantify successful invasion of Shigella flexneri. By using a series of polymerase chain reaction (PCR) and Gibson assemblies, we have constructed a plasmid with a RFP reporter to be expressed during successful invasion of Shigella spp. The IpaH9.8 MxiE promoter, which has been shown to be expressed upon cell entry, is integrated within the pUltra RFP plasmid and electroporated into a streptomycin-resistant S. flexneri strain. We will test the recombinant strain in vitro by activating ipaH9.8 in broth culture and inoculating a mammalian cell line (HCT-8). RFP expression will be monitored with a BioTek FLX-800 plate reader and visually imaged using a FX-EVOS microscope. The expected outcomes of this project will provide an accurate and efficient method of quantifying invasive S. flexneri. in vitro and in vivo, as well as quantifying pharmacodynamics and pharmacokinetics of new treatment therapies. The implications of this project are crucial to the advancement of shigellosis research and in furthering the efforts of the international community to abate the steadily increasing cases of drug resistant shigellosis cases observed worldwide.

Rheumatoid arthritis (RA) is an inflammatory autoimmune disease characterized by elevated inflammatory mediators, bone resorption, and cartilage destruction. These changes are caused in part by fibroblasts, which invade cartilage and amplify inflammation by producing a variety of cytokines and chemokines. Previous research suggests that activation of the receptor tyrosine kinases platelet-derived growth factor receptors (PDGFRs) may play a major role in fibroblast expansion and invasion in RA. These receptors are homo- or heterodimers composed of α or β subunits, and can be stimulated by five different ligands. Although the signaling pathways activated by PDGFR-α and PDGFR-β are very similar, they have been shown to play different roles in disease models. We propose that PDGFR-α and PDGFR-β activation produce independent responses in joint (synovial) fibroblasts, depending on both receptor signaling and ligand availability, contributing to their different roles in disease. We found activation of both receptors stimulated fibroblast proliferation and recycling of Cad-11, a cell-to-cell adhesion molecule that plays an important role in the signaling that produces the inflammatory response. This was found by comparing receptor phosphorylation data from Western Blot, ELISA, and flow cytometry experiments. However, when silencing PDGFRs with a multi-tyrosine kinase inhibitor, only PDGFR-α was found to affect Cad-11, meaning Cad-11 likely works through PDGFR-α to regulate cell proliferation. In contrast, PDGFR-β activation has been shown to induce a more motile morphology change in immunofluorescence experiments with 3D cell cultures. qRT-PCR experiments have also provided evidence that the same cell lines stimulated with different ligands resulted in distinct genotypes. These results support our hypothesis that PDGFR-α and PDGFR-β may have unique functions in synovial fibroblasts that contribute to RA pathology.

Mycobacterium tuberculosis (Mtb) is a bacterium that can cause both latent Mtb infection (LTBI) and tuberculosis disease (TB). LTBI affects one quarter of the world’s population of those who are LTBI positive, 5-10% develop TB disease which left untreated can be fatal. Most individuals who are highly exposed to Mtb develop LTBI and show positive results for the tuberculin skin test (TST) or interferon-g release assay (IGRA). The Hawn lab has followed a cohort of Ugandans with heavy exposure to Mtb within the household but test negative by the TST and IGRA tests and define these individuals as Mtb 'resisters'. The presence of these Mtb ‘resister’ genes suggests a possible pathway for host directed therapies. Using transcriptomic profiling to compare the ex vivo Mtb response in LTBI and resister monocytes, several candidate resister genes were identified. We hypothesized that these candidate resister genes modulate antimicrobial functions through an effect on pro-inflammatory cytokine secretion which restricts Mtb growth. Individual ‘resister’ genes were inactivated by CRISPR/Cas9 gene editing in THP-1 cells and then differentiated into macrophage-like cells prior to various stimulations. Macrophage signaling pathways were stimulated with LPS (Toll-like Receptor 4 (TLR4)), PAM3 (TLR2/1) as well as with whole cell lysate from Mtb. I measured pro-inflammatory cytokine secretion in supernatants after 24h (IL-6, IL-1b, and TNF-a). We identified two genes, SETDB1 and NFIL3_03, that may differentially regulate pro-inflammatory cytokines compared to the control THP-1 cells. By uncovering the function of these genes, we hope to better understand how Mtb infections can result in variable immunopathogenesis.

T-cell immunotherapy is a treatment utilising the body’s own immune system to target cancer cells. Dr. Marie Bleakley’s immunology lab at Fred Hutchinson Cancer Research Center uses T-cell immunotherapies to treat hematologic malignancies — blood cancers which include acute myeloid leukemia and myelodysplastic syndrome (MDS). These treatments rely on the identification of neoantigens (cancer-specific gene mutations) which are potentially highly cancer-specific and are promising as targets for new immunotherapies. My project will be in the identification of these neoantigens, under the supervision and mentorship of Dr. Melinda Biernacki, a research associate in Dr. Bleakley’s lab. My project will focus on generating in silico and preliminary in vitro data for candidate neoantigens in high-risk MDS.
The project will have two parts. First, the identification of candidate MDS neoantigens using human leukocyte antigen (HLA)-binding prediction algorithms to identify mutated amino acid sequences (peptides) that have a high probability of binding HLA molecules. Because T-cell antigens consist of a peptide-HLA molecule complex, this step will filter out mutant peptides that are unlikely to be recognized by T cells. Second, in vitro assays will determine which candidate neoantigens are immunogenic (can actually be recognized by the body’s T-cells). HLA-binding predictions do not reliably predict immunogenicity of candidate neoantigens, so in vitro testing is needed to assess T-cell recognition. The end product will be the identification of potential T-cell targets from mutations in various genes that are shared in subgroups of MDS patients that could be used to develop immunotherapies for MDS.

Interferons are signal proteins released by cells in response to infections which induce neighboring cells to synthesize anti-viral genes like Mx2, which encodes the MxB protein. MxB was thought to only restrict lentiviruses but recent studies have shown that MxB also restricts herpesviruses. It is hypothesized that herpesvirus and MxB have co-evolved in an ‘arms race’ to compete against each other. This suggests that regions of MxB that are under positive selection may have interfaces for its anti-herpesvirus activity. In order to test this hypothesis, I plan to determine whether different homologs of MxB can restrict human herpesviruses. I created MxB inducible cell lines using MxB homologs from three different primates - humans, rhesus monkeys and owl monkeys. Subsequently, I infected these cells with the following GFP labeled viruses: herpes simplex virus 1 (HSV-1), Kaposi’s sarcoma-associated herpesvirus (KSHV), and human cytomegalovirus (HCMV). I assessed the effect of these MxB alleles on viral replication by measuring viral titer and viral protein production (including GFP). I expect that the MxB restriction of herpesviruses will be species-specific, with only human MxB showing meaningful reduction in human herpesvirus replication. These results will guide future research aiming to understand the physical interactions and mechanisms of suppression between MxB and herpesviruses.

Social determinants of health (SDOH) are non-medical factors that contribute to a person’s health and well-being. The Centers for Medicare/Medicaid Services recently released a standardized 26-question survey designed to capture SDOH in the following 13 domains: living situation, food insecurity, transportation, utility help, personal safety, financial strain, employment, family and community support, education, physical activity, substance use, mental health, and disabilities. However, there is limited study using this survey tool in a safety-net institution. Harborview Medical Center (HMC) is the public hospital serving King County, WA and provides care to a wide variety of patients. This project was a prospective, observational study which aimed to investigate implementation of this survey tool and to measure relevant SDOH and other medical factors in this setting. My role included administering the survey to patients admitted to the HMC Hospital Medicine service from October to December, 2018. I collected data related to the duration of the survey and any challenges patients encountered responding to the survey, as well as review of patient charts for demographic information, medical system utilization, and medical history. In total, only 24 of 90 patients (27%) completed the survey. Major challenges included patient lack of interest, language barrier, ability to consent, and competing time demands. These results have implications for the generalizability of this tool and whether it comprehensively identifies SDOH needs.

Atherosclerosis, the underlying cause of most heart attacks and strokes, results from lipid accumulation in cells of the artery wall. Gene therapy, delivered directly to the artery wall, has the potential to prevent and reverse atherosclerosis. However, lipid accumulates primarily in cells below the endothelium, which are difficult to reach with gene therapy vectors. Our goal is to remove lipid from these cells by delivering therapeutic microRNA (miR) that increases cholesterol export from the cells. We hypothesized that if we introduced a therapeutic gene expressing the miR to endothelial cells (cells along the artery lumen), the endothelial cells would release this miR (anti-miR-33a-5p) via extracellular vesicles (exosomes) that transport miR between neighboring cells. We also hypothesized that smooth muscle cells (SMC) and macrophages would take up the therapeutic miR-containing exosomes, leading to higher expression of a critical cholesterol export protein (ABCA1) and increased cholesterol export. To test this in vitro, we introduced a therapeutic gene encoding the miR into endothelial cells and used RT-qPCR to test if the miR was released into the endothelial cell culture medium (CM) via exosomes. After confirming the presence of the therapeutic miR in exosomes purified from CM, we treated SMC and macrophages by incubating the cells with the exosome-containing CM. After incubation, we measured ABCA1 protein expression and cholesterol export. Expression of ABCA1 protein increased by 1.6- and 2.2-fold in SMC and macrophages, respectively, while cholesterol export increased by 1.4- and 1.6-fold. We conclude that gene therapy delivered to endothelial cells can produce therapeutic miR that is transferred to neighboring artery wall cells via exosomes, and increases cholesterol export in these target cells. If also effective in vivo, our approach has potential for reducing the severity of atherosclerosis by delivering therapeutic miR to cells that are difficult to reach with gene therapy.