Mycobacterium tuberculosis (Mtb) is a widespread disease affecting about 33% of the human population around the world. Treatment of tuberculosis (TB) is hampered by a need for lengthy, multi-drug regimens and drug resistance requiring development of novel therapeutics. Our laboratory is interested in harnessing the host immune response to enhance Mtb killing within the macrophage. Within the host, Mtb is found within lipid-laden macrophages where lipid droplets are hypothesized to both provide nutrients as well as sequester Mtb from intracellular killing mechanisms. Hydroxycarboxylic acid receptor 2 (HCA2) is a G-protein coupled receptor (GPCR) that maintains lipid droplets after activation by its ligands nicotinic acid (NA) and beta-hydroxybutyrate (BHB). In these studies, we assess NA, BHB, and the structurally related small molecules nicotinamide (NAM), sodium butyrate (NaB) and phenylbutyrate (PBA) modulate HCA2 signaling. We have transiently transfected CHO-K1 cells with plasmids encoding human HCA2 and aequorin, the latter which oxidizes coelenterazine in a reaction that generates a luminescent signal. The effective concentration (EC50) of each small molecule as a ligand for HCA2 can then be calculated from the luminescence curve. Preliminary work in our lab suggests that PBA inhibits Mtb replication within the host macrophage, but several mechanisms are likely involved. We hypothesize that NaB, PBA, and NAM will also modulate HCA2 activity given their similar structures to NA and BHB. Future studies will also interrogate whether this panel of small molecules impacts HCA2 gene expression, lipid droplet formation and Mtb replication in macrophages.

Current weight loss therapies are largely unsuccessful stressing the urgent need to develop new and effective strategies to treat the growing obesity epidemic. Oxytocin (OT) is a downstream target of the adiposity signal, leptin, and is an attractive therapeutic target to treat obesity in humans because it reduces body weight (BW) in diet-induced obese (DIO) rodents even in the face of an impaired ability of leptin to reduce food intake. Recent findings from our lab indicate that, while reductions in food intake contribute to OT’s ability to elicit prolonged weight loss in DIO rodents and nonhuman primates, mounting evidence implicates a role of increased energy expenditure (EE) through sympathetic nervous system (SNS) outflow to interscapular brown adipose tissue (IBAT) as contributing to weight loss. OT neurons in the parvocellular paraventricular nucleus are positioned to control SNS outflow to IBAT through either direct projections to the spinal cord or indirectly via the hindbrain nucleus of the solitary tract. Thus, these circuits are potential targets for pharmacological therapies to reverse the deleterious metabolic effects of central leptin resistance. We hypothesized that OT-induced stimulation of SNS outflow to IBAT contributes to its ability to elicit weight loss in DIO rodents. To test this, we determined the effect of exogenous fourth ventricular (4V) OT administration on IBAT temperature, BW, and body composition in DIO rats. We found that 4V OT increased IBAT temperature, reduced BW and body adiposity. Acute 4V administration of OT increased IBAT temperature during the test period relative to vehicle-treated controls (45 min measurement: vehicle: 37.2 ± 0.03 vs. OT: 38 ± 0.2°C; P<0.05). Collectively, these studies implicate a role for SNS outflow to IBAT in the mechanism by which OT increases EE and elicits BW loss in DIO rats.

Genetic variation plays an important role in an individual’s susceptibility to Mycobacterium tuberculosis. Common variation in the rs5743854 polymorphism was found to regulate Toll Interacting Protein (TOLLIP) mRNA levels in the TLR signal pathway in macrophages. Decreased TOLLIP mRNA was associated with an increase in inflammatory cytokines and decrease in disease-specific TCM cells, leading to increased tuberculosis susceptibility in adults. We are investigating the role of a long non-coding RNA, TOLLIP Antisense 1 (TOLLIP-AS1), which houses rs5743854 and is located immediately upstream of TOLLIP. We hypothesize that TOLLIP-AS1 regulates TOLLIP mRNA expression and therefore plays an important role in regulating immune responses during tuberculosis. We performed RT-PCR experiments using primary monocytes from multiple ethnic cohorts and plotted mRNA expression levels of TOLLIP against TOLLIP-AS1. We also generated overexpression and knockdown TOLLIP-AS1 cell and measured any subsequent effects on TOLLIP expression. The long non-coding RNA TOLLIP-AS1 may prove to be critical intermediate in regulating gene expression and provide new targets for TB host-directed therapeutics and vaccine adjuvants.

Hereditary Spastic Paraplegia (HSP) is a classification for a group of neurogenetic diseases that cause affected individuals to have contractions and stiffness in the limb muscles. This group includes a wide range of disorders that vary in age of onset, rate of progression, and severity. The purpose of my project is to identify gene variants involved in HSP and their causal mechanisms. My lab acquired DNA samples from affected and unaffected members of a family with an unassigned autosomal dominant HSP. To begin my analysis, we obtained whole exome sequencing on DNA from three affected members. In a file containing all the variants (differences from a reference exome) detected in any of these three subjects, I looked for previously identified causal variants to ensure that the family did not have a known HSP subtype. Then I chose candidate variants by filtering for those that were heterozygous in all the exome sequences, and ranked them based on their absence from the Exome Aggregation Consortium database, predicted change in the protein, relevance of the gene function, absence from the UW Center for Precision Diagnostics database, and dbNSFP model predicted impact. I selected a total of 50 candidate variants and am in the process of analyzing them further. I have amplified and sequenced DNA from all the family members to determine whether certain selected candidate variants are present in all those who are affected and absent from those who are not affected, since this is the pattern expected for an autosomal dominant condition. I have currently eliminated five candidate variants and expect to soon identify the causal variant. My project will contribute to our understanding of the pathogenesis of HSP and improve clinical diagnostics for this disorder. The implications of my research could extend to other disorders if a novel mechanism was elucidated.

 Oncolytic virotherapy is a cancer therapy that uses a modified virus to attack and kill cancer cells. These viruses can naturally only replicate in cells that lack a functional p53 gene, a gene that normally regulates the cell cycle and prevents cancer. To replicate, many of these viruses disable the p53 gene themselves, so that they can reproduce in healthy cells. However, by interrupting the processes used to disrupt the gene, virologists have engineered viruses that can only reproduce in, and kill cancer cells. One limitation of this process is that the patient’s innate immune system can prevent virus replication in cancer cells that naturally produce defensins - a class of small antimicrobial proteins. My lab has previously shown that one of these, synthetic Human Defensin 5 (HD5), blocks replication of Adenovirus 3, which is one virus used for oncolytic virotherapy. It would be important to understand in which types of cancers HD5 is produced, so that virologists can better design oncolytic viruses to bypass HD5. My research was the first demonstration that HD5 is secreted and upregulated in its active form by epithelial ovarian and lung cancer cells in situ. This makes the cancer cells especially hard for the virus to kill. To demonstrate this, I stained sections biopsies of a wide array of human tumors using a mouse anti-HD5 antibody. There was a clear difference in the staining of ovarian and lung cancer samples and their respective controls with no anti-HD5 antibody. This is especially significant because so little is known about what factors contribute to the effectiveness of oncolytic virotherapy.

Depression, anxiety, and other manifestations of distress are major causes of morbidity for cancer patients and contribute to a decreased quality of life. A combination of pharmacological, behavioral, and psychiatric interventions have been shown to lessen the symptoms of depression and improve patients’ quality of life. The Integrated Psychosocial Oncology Program (IPOP) at the Seattle Cancer Care Alliance (SCCA) uses the collaborative care model to provide cancer patients with care and support to manage symptoms of distress while receiving treatment at the SCCA. This evaluation of the SCCA IPOP program aims to 1) illuminate barriers and risk-factors in the pathway to psychosocial services, including screening, referrals, and follow-ups; and 2) develop process interventions to improve outcomes in treatment. Preliminary analysis shows that of the patients referred to the IPOP between June 2015 and May 2016 (N=919), 83.2% were screened for depression and anxiety, but only 28.3% (N=440) of those found to demonstrate moderate symptoms of depression or anxiety received a follow-up screening. Without follow-up screenings, the SCCA is unable to evaluate the efficacy of its interventions provided by the social work team. In order to improve the frequency of these follow-ups, the SCCA Social Work team is introducing a patient roadmap to improve patient engagement and changing data-collection processes to improve the availability of population health metrics. These changes are expected to improve two-month follow-ups, and improve the outcomes of SCCA social work patients, which will be monitored over the next 6 months.

Adverse Events (AEs) are detrimental medical incidents in a research participant that may or may not be related to the investigational agent. During clinical trials, physicians are required to document attribution of AEs with limited guidance regarding how to assign attribution. The objective of this study was to determine the accuracy of physician based attributions for AEs occurring in cystic fibrosis (CF) patients participating in blinded randomized controlled trials (RCTs). This retrospective study pooled clinical trial data from 4 CF RCTs; all participants who experienced ≥1 AE were included. A repeated measures logistic regression model using generalized estimating equations and adjusted for baseline demographic variables (gender, age, CF genotype), comorbidities, and lung function (forced expiratory volume in one second; FEV1). The primary predictor of interest was receipt of study drug versus placebo. The primary outcome of interest was physicians’ determination of the AE as related to study drug. Study patients were young with mild lung disease. Receipt of study drug was not significantly associated with the likelihood a physician would deem an AE to be related to study drug. However, we found significant associations between age, gender, and FEV1 and our outcome of interest– presence of an AE felt by a physician to be related to study drug. These findings suggest a biased assessment of physician attribution. Physician assessment of relatedness in RCT’s is a key requirement per the Food and Drug Administration (FDA). We found no significant association between AEs coded as related to study drug and actual study drug receipt. Interestingly, we found other associations implying an intrinsic bias in physician assessment based on age, gender and disease severity. Further research may clarify whether changes are needed to current FDA guidelines.

Patients undergoing ventricular assist device (VAD) therapy are by definition very sick and typically told that they have a high likelihood of dying in the next year without VAD therapy. Though most patients have improved functional status and quality of life, repeated hospitalizations and potentially catastrophic complications post implant can be burdensome. The research aims to assess for decisional regret or ambivalence about VAD implant. During a 3 year program all post VAD implant patients were referred to palliative care. Patients were administered a 5 question decision regret survey that correlates well with decision satisfaction and decisional conflict. On a scale of 0-100, scores >25 were considered to reflect significant ambivalence, and scores between 10-25 were defined as mild ambivalence. Patients were also administered the PHQ-9, a tool for depression inventory. The results found that 102/129 (79%) patients completed decision regret surveys. 64/102 (63%) scored 0, 7/102 (7%) scored >25, and 29/102 (28%) scored between 10-25. The average age of the patients scoring >25 was 44, compared to 55 of the whole cohort. Of those that expressed some degree of ambivalence (n=36), 11/36 (31%) met criteria for at least moderate depression, compared with 18% for the whole cohort. 26% of the total cohort had VADs as bridge to transplant (BTT), while 13/29 (44%) ambivalent patients and 4/7 (57%) very ambivalent patients were living with their VAD as BTT. A majority of patients do not regret their decision; however, a significant minority expressed mild ambivalence, and a small number expressed significant ambivalence. Ambivalence was more common in those who are younger, those with VAD as BTT, and in patients with depression. It is important to recognize pre-implant that patients have different decision-making needs, and that managing their expectations may be a key component of helping them to adjust post VAD.

Human immunodeficiency virus (HIV) infection is a global health issue and consists of two main types, HIV-1 and HIV-2. As the backbone of all first-line treatments of HIV, nucleoside reverse transcriptase inhibitors (NRTI) have a critical role in antiretroviral therapy. EFdA (4´-ethynyl-2-fluoro-2´-deoxyadenosine; MK-8591; Merck & Co.) is an investigational NRTI that blocks HIV-1 replication in culture with 50% effective concentrations (EC₅₀) in the low nanomolar to picomolar range. However, studies evaluating the activity of EFdA against HIV-2 and against NRTI-resistant mutants of HIV-2 are lacking. As a result, HIV-1 and HIV-2 isolates from antiretroviral-naïve individuals were tested against EFdA in single-cycle infections of a human cell line (MAGIC-5A cells). Site-directed mutants of HIV-2 reverse transcriptase were generated in a full-length plasmid clone and were evaluated for EFdA resistance in the single-cycle assay. EFdA inhibited HIV-2 infection of MAGIC-5A cells with mean EC₅₀ values (± SD) of 0.55 ± 0.17 nM for 8 HIV-2 group A isolates and 0.50 ± 0.12 nM for 6 HIV-2 group B isolates (range = 0.25–0.76 nM for all 14 HIV-2 strains tested). In contrast, the mean EC₅₀ for 10 HIV-1 isolates, including group M (subtype A, B, C, D and F) and group O isolates, was 2.56 ± 0.56 nM (range = 1.96–3.78 nM; p<.0001 for HIV-1 versus HIV-2, Welch’s t-test). EFdA is the most potent inhibitor of HIV-2 replication described to date and is more active against HIV-2 than against HIV-1 in culture. EFdA also inhibits multi-NRTI–resistant HIV-2 mutants with single-cycle EC₅₀ values <12 nM. Our data indicate that EFdA should be evaluated in clinical studies involving HIV-2-infected individuals.

Two human retroviruses cause AIDS: HIV-1 and HIV-2. HIV-2 is less pathogenic than HIV-1, with lower plasma viral loads, decreased transmission and mortality rates, and a slower decline in CD4+ T cells. Despite this, many patients progress to clinical AIDS and may benefit from antiretroviral therapy (ART). HIV-2–infected patients failing first-line ART often harbor drug-resistant strains of the virus that limit second-line treatment. Consequently, there is a need for rapid, inexpensive HIV-2 drug resistance testing that can guide second-line therapy. In this study, we optimized and validated a novel genotypic drug resistance test for HIV-2 using dried blood spots (DBS). Specimens for this project were collected as a part of an ongoing cohort study of ART for HIV-2 infection in Senegal, West Africa. Blood samples from HIV-2–infected patients were spotted onto filter paper and shipped via courier “overnight” to Seattle at ambient temperature. We then extracted viral RNA and DNA from the DBS, amplified genes of interest using nested PCR, and identified mutations at major drug resistance sites after population sequencing. After obtaining genotypes from only three of 42 (7%) of DBS from a previous protocol, we designed a new, optimized protocol and were able to genotype 10 of 22 (45%) DBS, including five of seven (71%) with HIV-2 plasma viral loads over 50 copies/mL. Drug resistance mutations V47A (protease) and M184V (reverse transcriptase; RT) were observed in 63% and 75% of genotypes, respectively – 75% had evidence of multi-class resistance to both protease and RT inhibitors. These results show that DBS genotyping can identify major drug resistance mutations in HIV-2 patient samples, thereby guiding second-line ART for HIV-2 infection. Because DBS-based resistance testing is a low-cost testing approach which provides results within a clinically-actionable timeframe, we expect that this innovation will improve HIV-2 patient care in resource-limited settings.

Merkel cell carcinoma (MCC) is a rare, aggressive neuroendocrine skin cancer, which primarily afflicts Caucasian and elderly patients. While MCC patients with intact immune systems have a 20-30% risk of recurrent MCC within 2 years of diagnosis, risk of recurrence or death is higher among immunosuppressed patients. However, it is unknown whether immunosuppression type affects progression-free and disease-specific survival. From a large data repository, we identified and retrospectively evaluated 95 MCC patients that were chronically immunosuppressed at the time of diagnosis and were treated with curative intent between 1988 and 2016. There were five types of chronic immunosuppression represented: chronic lymphocytic leukemia (CLL, 29% of immunosuppressed cases), solid organ transplant (SOT, 23%), autoimmune disorders (AD, 22%), other hematologic malignancies (OHM, 17%), and HIV/AIDS (9%). Progression free survival (PFS) and disease specific survival (DSS) were estimated by the Kaplan-Meier method. As part of curative-intent therapy, the majority of patients were treated with wide local excision (89%) followed by adjuvant radiation (82%). Immunosuppression was associated with poor outcomes among MCC patients: 72% of such MCC patients experienced recurrent disease at a median time of 7.4 months after diagnosis (range, 1-75 months). 45% of recurrences in immunosuppressed patients were distant, while 30% were regional and 25% local/in-transit. 2 yr-PFS and DSS by immunosuppression type was: CLL (PFS 20%/DSS 59%), SOT (0%/32%), AD (14%/52%), OHM (29%/43%), and HIV/AIDS (0%/0%). Immunosuppression is associated with a high risk of recurrence and mortality in MCC patients. While these results are preliminary, they suggest that the type of chronic immunosuppression may affect outcomes in MCC patients.

Conjugated linoleic acid (CLA), the only naturally-occurring trans-fat, is a fatty acid that is known to promote weight loss and increase energy expenditure. While it is considered a potential treatment for those struggling with weight-induced illness, previous research has shown its association with impaired glucose metabolism and inflammation. Using qRT-PCR to analyze expression of genes important for glucose metabolism and inflammation, we studied whether rosiglitazone, an insulin-sensitizing agent, improves CLA-mediated insulin resistance and adipose tissue inflammation in obese mice. We have previously examined epididymal and inguinal white adipose tissues and found that rosiglitazone increased expression of genes important for insulin responsiveness (such as adiponectin and PPARG), but decreased genes associated with inflammation (such as Mac2). In this study, we assessed the same gene expression profiles in mesenteric and retroperitoneal white adipose tissues, two adipose tissue depots that are understudied, but that could be important sites of CLA and/or rosiglitazone metabolism. Rosiglitazone effectively increased insulin sensitivity in mice with CLA-mediated insulin resistance, as made apparent by improved glucose and insulin tolerance tests. Further, rosiglitazone improved CLA-mediated decreases in the insulin sensitizing hormone adiponectin, measured in plasma and in both mesenteric and retroperitoneal white adipose tissues. Rosiglitazone had no effect on adipose tissue inflammation, however. This study demonstrates possible methods to improve CLA-induced weight loss by increasing insulin sensitivity with rosiglitazone treatment, and provides a possible intervention for those struggling with weight-related disease.

A glomerulus is a structure in the kidneys that filters the blood in the first step of urine production. Focal segmental glomerulosclerosis (FSGS) is a condition where the glomeruli in the kidneys lose podocytes (epithelial cells lining the blood capillaries), severely limiting their ability to filter blood and leading to potentially fatal kidney diseases such as chronic kidney disease (CKD). One way that the body responds is through mesenchymal-to-epithelial transformation (MET), where mesenchymal juxtaglomerular (JG) cells, identifiable by renin expression, migrate into the glomeruli to replace lost podocytes. My project assesses the role of WT-1, a gene linked to the differentiation of cells in the kidney, in MET of cells of renin lineage (CoRL) in FSGS. WT-1 knockout and wild-type mice were used, and FSGS was induced by injecting anti-glomeruli IgG. Some of the mice were treated with the drug Enalapril each day after FSGS was induced, which has been shown to increase the number of CoRL. Baseline tissue was collected via a kidney biopsy one week before FSGS was induced, and a kidney necropsy was carried out 28 days after FSGS was induced. I then stained kidney sections for mesenchymal and epithelial markers using indirect fluorescent immunohistochemistry. From visual analyses of the stained kidney sections, we observed that in wild-type kidneys with FSGS, mesenchymal marker expression in CoRL generally decreased and epithelial marker expression in CoRL generally increased relative to baseline biopsies. Moreover, expression of all markers in WT-1 knockout kidneys with FSGS remained similar to baseline expression. We concluded that WT-1 is necessary for MET of CoRL in FSGS. These results highlight the importance of considering WT-1 in podocyte regeneration in experimental FSGS, bringing us one step closer to effectively treating the 5400+ people diagnosed with this disease each year.

The accumulation of pathological tau protein in the brain neurons occurs in many neurodegenerative diseases. Alzheimer’s disease and related dementia disorders are the most common diseases with pathological tau. In order to identify potential therapeutics for Alzheimer’s disease and related disorders, I screened the National Institute of Health’s Clinical Compound Library for modifiers of the disease associated phenotype seen in tau transgenic Caenorhabditis elegans (C. elegans). The compound library consists of drugs with a history of use in humans that we can assess for other potential uses, a process known as drug repurposing. Observing C. elegans disease models for specific drug effects may ultimately lead to identifying ways to alter or prevent accumulation of tau aggregates in the brain. Populations of tau-expressing C. elegans were cultured on nematode growth media (NGM) in the presence of a single library compound and assessed for modification of disease phenotype at day one of adulthood; roughly 3 days after eggs are placed on the media. I specifically looked for improvement or deterioration of coordination as well as the speed of growth, number of offspring, mortality, and general health and size of the population as compared to control populations without drug. I then evaluated the data and selected the best compounds for further analysis on the basis of each compounds dose responsiveness. Drugs generally have optimal effect at a specific dosage, so by observing a range of concentrations of the drug I can determine the best concentration for study. Once this has been completed, I will analyze the hits more closely and follow up with further experimentation including quantitative analysis of C. elegans behavioral response, and tau protein biochemistry. We hope that this work may be able to identify potential therapeutics able to ameliorate symptoms in dementia patients with pathological tau.

Chronic kidney disease currently has no cure but continues to plague our population, demanding new effective strategies to treat kidney disease through the study of biochemical mechanisms of fibrosis. Kidney fibrosis begins with the loss of function of parenchymal cells that leads to excessive scarring in the kidney due to excessive extracellular matrix formation. Several studies in both human and experimental mice models have shown that increased oxidative stress plays a critical role in renal fibrosis due to the generation of reactive oxygen species. However, the mechanisms by which oxidative stress promotes kidney injury or fibrosis remains unclear. PRX2 is an antioxidant enzyme that normally protects the cell against oxidative stress by the reduction of hydrogen peroxide and oxidized proteins. Normally, an increase in oxidative stress has shown to increase an inflammatory response, potentially leading to fibrosis. Unfortunately, the exact mechanisms by which this occurs is unclear. In contrast to its role as an antioxidant, recent studies suggest that PRX2 may be an important mediator of the proinflammatory response as a direct result of oxidant stress. Based on these studies and our preliminary data, the Okamura Lab currently hypothesizes that the loss of PRX2 leads to a significant decrease in renal fibrosis by attenuating the inflammatory response. Our preliminary data suggests that PRX2 is expressed in tubular epithelial cells in normal kidneys and expression decreases with progressive injury by unilateral ureteral obstruction (UUO). Despite the decrease in PRX2 during UUO, PRX2-deficient mice had a significant reduction in fibrosis with a significant decrease in the proinflammatory cytokine TNF-alpha. Methods to further study the mechanism of PRX2 include western blotting, immunostaining, and quantitative PCR. As this study continues, the information on PRX2 will be a valuable asset to developing antioxidant treatment therapies to find a cure for chronic kidney disease.

Currently, the only two treatments available to people with chronic kidney disease are organ transplants and dialysis. With such a high demand for kidneys, regenerative medicine is needed to create new therapies. Human pluripotent stem cells (hPSCs) can be used to generate immunocompatible tissues on-demand, which can be used to study human disease processes or potentially transplanted back into the original patient as a tissue replacement therapy. New protocols allow the differentiation of pluripotent stem cells into small, kidney-like organoids that contain the major proximal structures of the nephron, the functional subunit of the kidney. These organoids include podocytes, proximal tubules, distal tubules, and endothelial cells, but they lack a collecting duct system, a crucial component of the kidney. To address this need, we have developed a protocol to differentiate hPSCs into ureteric bud (UB) cells, which are the precursors of collecting duct cells. Undifferentiated hPSCs were treated with a growth factor, glial cell line-derived neurotropic factor (GDNF), that signals the outgrowth of the UB in the human kidney, and a differentiation factor, CHIR 99021, at different concentrations. Immunofluorescence was used to characterize the resulting cells, which were stained with Dolichos biflorus agglutinin (DBA), a collecting duct marker, DAPI, a DNA marker, and GATA 4, a transcription factor that is important for kidney development. We found that both DBA and GATA 4 were co-expressed together in isolated patches of cells under one of the conditions tested. This staining pattern suggested that these were UB cells. This is the first time UB cells have been differentiated from hPSCs. The development of this differentiation protocol will lead to the growth of the collecting duct system in the kidney organoids, which will optimize them for kidney disease modeling, high-throughput drug screening, and regenerative medicine approaches to reduce the need for kidney transplants.

The goal of this research is the generation of induced pluripotent stem (iPS) cells and derived kidney organoids from urinary cells. iPS cells are somatic cells reprogrammed to an undifferentiated state, making them an exciting advancement in regenerative medicine as they could model an in-vitro progression of many disorders and show great promise for replacing or repairing tissue damaged due to disease or injury. To generate new kidney tissues, adult somatic cells were isolated from a patient’s urine sample. This method of sample collection is advantageous over the traditional skin biopsy because it does not require a physician and is easier for the patient to supply. This protocol was further optimized to accommodate remotely collected urine samples, showing the ability to isolate somatic cells from patients located across the globe. We successfully isolated somatic cells with various morphologies, ranging from epithelial to mesenchymal-like cells, from 25 different patients. By introducing into these cells RNA encoding four specific transcription factors, a safer alternative to introducing DNA as it eliminates the risk of inserting a mutation into the patient’s genome, we successfully reprogrammed urinary cells of an international patient with autosomal recessive polycystic kidney disease into iPS cells. These iPS cells were further differentiated into kidney organoids, which have the capacity to become a powerful tool in improving current therapies for kidney disease as an in-vitro model. Thus, for the first time, we have generated patient-specific kidney tissue from a urine sample. This will increase our understanding of the disease and improve current methods for the screening of investigational therapeutics. The ability to create new kidney tissues in the lab also raises the possibility of transplanting these tissues back into their original patients, where they could potentially function without the need for immunosuppression.

Store-and-Forward Teledermatology is used as a system for increasing access to dermatology care for patients in the Veterans Health Administration (VA). Where dermatologists are not available, primary care clinics partner with teledermatology to help patients access high-quality, timely healthcare. Rather than being seen face-to-face by a dermatologist, patients’ skin lesions are imaged and sent asynchronously to a teledermatology reader (a dermatologist) in another location. Many types of skin conditions can be diagnosed through this system, including melanoma, a dangerous form of skin cancer. While this health system has demonstrated good diagnostic sensitivity for melanoma, little is known about its positive predictive value (PPV), also called precision. PPV is the proportion of melanoma diagnoses that are confirmed by pathology to be truly melanoma. Pathology diagnosis is considered the ‘gold standard’ for diagnosing dermatological conditions. Because the threat of melanoma is taken very seriously, low PPV leads to more unnecessary biopsy procedures and pathology consultations- actions that strain VA health system resources. This quality improvement study investigated the PPV of all teledermatology readers in the Seattle VA program from February 2015 – January 2016. Chart reviews (n=552) were completed and data were analyzed to determine an overall PPV rating, to find individual provider PPV ratings, and to identify factors correlated with high PPV. We hope to use this analysis for two primary quality improvement actions. First, we aim to develop PPV standards for teledermatology providers to meet. Secondly, we aim to identify what steps could be taken to maximize teledermatology reader PPV. Policy changes could be applied to help minimize waste in our teledermatology program.

Duchenne Muscular Dystrophy (DMD) is an X linked, recessive genetic disease caused by mutations in the dystrophin gene. The disease is characterized by progressive muscle degeneration. Dystrophic mice (mdx^4cv) are a DMD animal model. Here, we compared mdx^4cv and WT mice to quantitatively assess motor performance. We hypothesized that the mdx^4cv mice would show differences in gait compared to WT as a result of muscle weakening. To test our hypothesis, mice were assessed at 1.5, 3 and 6 months of age using the Noldus CatWalk XT system to analyze gait. The mice voluntarily moved across an enclosed walkway on a backlit glass plate. The prints were captured by a camera and later analyzed using the CatWalk software. Our results show that there are significant differences in gait between the WT and mdx^4cv mice. The mdx^4cv mice were slower to cross the runway than WT at 1.5 (mdx^4cv: 3.60.8s WT: 2.80.5s) and 3 months (mdx^4cv: 3.80.9s, WT 2.51.2s ). The distance between the front paws of the mdx^4cv mice were wider than WT at 1.5 months (mdx^4cv: 3.60.8cm WT: 2.80.5cm), 3 months (mdx^4cv: 3.80.9cm WT: 2.51.2cm) and 6 months (mdx^4cv: 1.50.2cm WT: 1.20.2cm). Print position is the distance between the position of the hind paw and the position of the previously placed front paw on the same side. In mdx^4cv mice the print position was larger than WT at 1.5 (mdx^4cv: 1.30.8cm WT: 0.40.2cm) and 3 months (mdx^4cv: 1.40.8cm WT: 0.50.4cm), indicating that the hind leg moved a shorter distance in a single stride. The gait analysis assay can be extended to assess gene therapy based treatments that our lab is currently developing. This will allow us to determine if the treatments are successfully able to mitigate the effects of DMD.

Studies using primary airway epithelial cells from asthmatic and healthy donors have reported differences in gene expression of a variety of genes between cells from asthmatic and healthy donors. However, few studies have investigated the stability of gene expression by primary cells with increasing cell passage number, or how viral infection might affect the stability of gene expression with increasing cell passage number. This research investigated: 1) whether expression by primary bronchial epithelial cells (BEC) of genes IFIH1, which codes for a viral sensor protein with an important role in the innate immune response, and TGFβ2, a pro-remodeling cytokine, from asthmatic and healthy children, is stable with increasing cell passage number, and 2) if BEC infection with respiratory syncytial virus (RSV) alters the stability of IFIH1 and TGFβ2 expression with increasing cell passage number. To answer these questions, we differentiated passage 1, 2, 3, 4, and 5 BECs from asthmatic and healthy children at an air-liquid interface for 3 weeks. RNA was then harvested from BECs and RT-PCR was performed for TGFβ2 and IFIH1. To assess the expression stability across increasing cell passage number for each gene, we compared expression at passages 2-5 to expression at passage 1. Preliminary data collected shows that expression of TGFβ2 and IFIH1 from asthmatic and healthy children was stable with no significant differences between passages 1, 2 and 3. However, expression at cell passages 4 and 5 was significantly greater and more variable than by passage 1 BECs. We anticipate gene expression to be even more variable for RSV-infected passage 4 and 5 BECs. These observations illustrate the importance of using BECs from passage ≤ 3 when comparing gene expression between asthmatic and healthy primary BECs, and of characterizing the expression pattern across increasing cell passage number for each gene studied.

Mammalian cardiac myocytes (CMs) decrease proliferation soon after birth. This is caused by silencing of cell cycle gene expression. Gene activity is epigenetically regulated by physically changing chromatin structure. Generally, gene silencing is mediated by heterochromatin because of its tightly packed structure, which limits accessibility for transcription factors to bind to the gene promoters. Silenced chromatin is believed to form when heterochromatin protein 1 (HP1) binds to histone H3 lysine 9 tri-methylation (H3K9me3) to condense chromatin into heterochromatin. We previously found that demethylation of H3K9me3 inhibited cell cycle gene silencing in CMs and resulted in hyperplastic heart growth. This suggests that H3K9me3/HP1 pathway is an important regulator for cell cycle gene silencing. There are three isoforms of HP1 (alpha, beta and gamma) expressed in mammals, but we found that HP1 gamma was most highly expressed in CMs. Therefore, we created a cardiac specific HP1 gamma knockout (KO) mouse and tested our hypothesis that loss of HP1 gamma would reactivate cell cycle gene expression and induces cardiac growth. Using RT-PCR we confirmed deletion of critical exons of HP1 gamma mRNA. Western blot demonstrated significant reduction of HP1 gamma protein in KO CMs. Immunofluorescence staining showed HP1 gamma expression was reduced specifically in CMs. HP1 gamma KO mice showed normal heart growth and no significant difference in heart mass was observed compared to control hearts. Consistent with this result, there were no significant differences in cell cycle or cardiac gene expression between HP1 gamma KO and control CMs. Interestingly we found an increase in protein level of HP1 beta, but not HP1 alpha, in HP1 gamma KO CMs. These data demonstrate that HP1 gamma’s function is redundant in cell cycle gene silencing and cardiac growth, which could be due to HP1 beta compensating for the loss of HP1 gamma in CMs.

We demonstrate that results from genetic association studies of telomere length performed in European populations are not generalizable to African Americans. Telomeres are repeated segments of DNA at the ends of chromosomes important for maintaining healthy chromosomes. Telomeres have been associated with a number of diseases and age-related conditions, however this relationship is complex because it is uncertain if an individual’s telomere length increases their risk for disease, or if disease and other environmental exposures are modifying the individual’s telomere length. Recent studies have scanned the entire genome for genetic variants that may be associated with telomere length. Nine highly significant genetic variants associated with telomere length have been identified, though the study populations have been of exclusively European descent. Since it is well known that the genetic architecture varies extensively across racial populations, we examined whether the nine genetic variants were associated with telomere length in 1,442 African American children (age 8-21) from the Study of African Americans, Asthma, Genes and Environments. We examined the genotypes for each variant using multivariable linear regression to test whether the nine genotyped variants were associated with telomere length. We found that the nine variants identified in European populations were not significantly associated with telomere length in African American children. In addition to these nine variants, we examined locations across the entire genome for association with telomere length. We found several genetic loci associated with telomere length that were specific to sex and asthma status. These loci map to chromosome positions 6p22.1, 13q21.1, 20p11.23 and genes INPPB4 and CFTR. To our knowledge, this is the first study demonstrating that the nine variants associated with telomere length in European populations are not generalizable to African Americans. Our study underscores the necessity for including diverse populations in biomedical research.

Sirtuins are histone deacetylases- enzymes that remove acetyl and various acyl groups from a lysine amino acid side chains on histone and other client proteins. Sirtuins are NAD+ dependent, meaning that they function in the presence of the oxidized form of the coenzyme NAD. When an acyl group is removed, the resultant positively charged protein has a stronger affinity for the negatively charged DNA phosphate backbone. As a result, DNA wraps more tightly around the histone and transcription is repressed due to closed chromatin. Along with chromatin modification, sirtuins modulate various cellular processes through deacetylation of other clients. There are seven types of sirtuins, of which Sirtuin 2 (SIRT2) is the main focus of my research.  The goal is to identify compounds that inhibit SIRT2 activity, while have minimum inhibition to structurally similar to SIRT1. Compounds are initially screened at 50 μM to test for high inhibition of SIRT2 and low inhibition of SIRT1. Compounds that inhibit SIRT2 activity by more than 90% are then evaluated for a dose response curve in order to determine the IC50 of each compound, or the concentration at which the enzymatic activity is reduced to 50%. The last stage of testing in my research is to test the compounds on various mammalian cancer cell lines. We found that Burkitt’s lymphoma derived cell lines are most sensitive to SIRT2 inhibition, giving IC50 values in the <10 μM range. My research will contribute to the ongoing search for Sirtuin2-inhibiting compounds for the treatment of B-cell malignancies.

Respiratory syncytial virus infection (RSV) is the most common cause of hospitalization in infants in the United States. It has now been increasingly recognized to cause disease in elderly adults and those with chronic lung disease. We aim to identify risk factors associated with hospitalization due to RSV, severity of infection, and the neutralizing antibody response to infection in adults acutely infected with RSV. Patients admitted to Harborview Medical Center (HMC) with RSV are enrolled and given a brief questionnaire about their family and home, health history, and their drug use. In addition, serum samples are collected and tested for neutralizing antibody response to RSV. Analyzing the titers from the neutralizing antibody test identifies patients with a higher antibody response to infection. This can lead to a better understanding of the characteristics associated with more virulent RSV symptoms. With these data we will be able to better understand the immune response to infection as well as other factors that may play a role in disease severity due to RSV.

Alzheimer’s Disease (AD) is a debilitating neurodegenerative disease that currently affects over 5 million Americans. We have previously demonstrated genetic and neuropathology-based variation across cognitively-defined AD subgroups. Here we sought to determine whether patterns of vascular risk factors differed across cognitively defined subgroups. We used data from the Seattle-based Adult Changes in Thought (ACT) prospective cohort study of individuals age 65+ and dementia-free at enrollment. ACT follows people at 2-year intervals to identify incident dementia and AD. We used cognitive data at the time of AD diagnosis to determine scores for memory, visuospatial abilities, language, and executive functioning. We used these scores to determine each individual’s average cognition and then domain-specific deficits below that individual average. We used these data to define six groups: no prominent domain, memory-, visuospatial-, language-, or executive functioning-prominent. We evaluated risk factors for these six groups from self-reported medical conditions including diabetes, stroke, and hypertension, and diagnosed atrial fibrillation or coronary artery disease. We used multinomial logistic regression models with the no prominent domain group as the reference. To account for multiple comparisons, we present tests of the null that each risk factor is unrelated to each subgroup, and an omnibus test of the association between each risk factor and any subgroup. Of 825 cases, nearly half had no prominent domain, a few had multiple prominent domains, and a single domain was prominent for the remainder. Memory-prominent domain was more common in people with diabetes and executive functioning-prominent was less common, although the omnibus test did not reach statistical significance (p=0.08). Despite low statistical power due to small group sizes, our results suggest possible differences in risk associated with diabetes. Subsequent work will refine these investigations using medications and evaluating glucose levels over time. These results support additional efforts to further understand cognitively-defined AD subgroups

Preliminary work has revealed genetic heterogeneity among Alzheimer’s disease (AD) subtypes defined by cognitive domain-specific impairments. There are currently no reports of risk factor associations for these AD subgroups. We used cognitive data from 825 Adult Changes in Thought (ACT) participants at the time they were identified with probable or possible AD to generate scores for memory, executive functioning/attention, language, and visuospatial ability. We determined individual mean scores across all domains, and identified specific impairments as >0.75 SD below each individual’s mean domain score. Depressive symptom severity was measured with the CES-D, and study staff collected self-reported data on traumatic brain injury (TBI) exposure. We used multinomial logistic regression with the “no prominent domain group” designated as the reference category. We determined risk ratios for depression and TBI for each subgroup vs. the no prominent domain subgroup, and tested significance of any heterogeneity with an omnibus test. We controlled for age, sex, APOE genotype, and years of education. Education's role as a risk factor varied across the five cognitively-defined AD subgroups (omnibus p=0.04). Education predicted development of memory prominent AD rather than AD with no prominent domain (risk ratio [RR] = 1.08 per year of education, 95% confidence interval (CI) 1.00, 1.16, p=0.05; omnibus p=0.04). Depression also varied across AD subgroups (omnibus p = 0.04). Higher CESD scores were associated with lower risk of developing memory prominent AD compared to no prominent domain AD (RR = 0.93 per point on the CESD, 95% CI 0.88, 0.98, p=0.01). We did not find differences in risk associated with TBI. Cognitively-defined AD subtypes show heterogeneity in their associations with depressive symptoms and education. It is possible that these risk factors are associated with biological differences across subgroups.

In resource-limited settings, premature and small for gestational age (SGA) births are common and increase risk of infant mortality. During pregnancy, exposure to high pathogen levels and other health risks increases inflammation and may result in premature or SGA infants. Cytokines are small secreted proteins involved in the inflammatory immune response. This experiment examines variations in 25 cytokine levels in sera collected from pregnant women during the second trimester of gestation and again at the time of delivery. Samples selected from women who delivered full term SGA (n=50), preterm SGA (n=50), preterm appropriate for gestational age (AGA) (n=50), and full-term, AGA (n=50) infants were tested using a Luminex 25-Plex Magnetic Assay to compare cytokine level changes in these populations. I plan to conduct and analyze this assay which has not yet been completed, but preliminary data shows we should expect elevated levels of cytokines in those mothers with preterm and SGA infants. Overall this experiment provides a better understanding of cytokine profiles unique to preterm and SGA infants in resource poor conditions, and could provide novel therapeutic targets for the prevention of infant mortality associated with these conditions.

Age related changes in brain composition contribute to cognitive and functional decline. It is established that there is a decrease in vascularity, neuron density and brain volume, but changes in the extracellular matrix (ECM) components that provide support to microvasculature and cellular networks are understudied. The brain ECM is comprised primarily of Hyaluronan (HA) and HA-associated ECM, such as chondroitin sulphate proteoglycans. HA is a non-sulfated glycosaminoglycan that is synthesized at high molecular weight, but readily degraded to low molecular weight HA that is neuroinflammatory and damaging to microvasculature. We have previously shown that brain microvascular density decreases with aging and that this decrease is associated with increased HA accumulation. We now examine regional localization of HA, HA-associated ECM and the brain microvasculature in aging. Young (4 months, n=15), Middle (14 months, n=5) and Aged (24-25 months, n=15) murine brains were examined. Mouse brain sections were labeled with biotinylated HA binding protein or an antibody against chrondroitin sulfate proteoglycans. Brain microvasculature was identified by labeling with solanum. The cerebral cortex, midbrain, forebrain, cerebellum, and striatum were imaged (a minimum of 3 sections per region) at 10x. The location, association with microvasculature, and intensity of staining were quantified by ImageJ. We find that with aging, there is an increase in HA throughout all regions of the brain that is most prominent in the cortex. In contrast, there were no significant differences in CSPG accumulation with aging in any of the regions examined. We propose that the increase in HA, without a concomitant increase in HA- associated ECM to provide stability, leads to greater amounts of neuroinflammatory low molecular weight HA in aged brains. We postulate these changes promote loss of microvascular density, which contributes to other deleterious changes in the brain with aging.

Discussing end-of-life care, especially for patients with serious illness, is an important way to ensure that clinicians understand their patients’ wishes and that patients therefore receive the types of care and treatments they want. Conversations about end-of-life care are often not a part of outpatient clinic visits even though the outpatient setting is the best place to have these conversations before a crisis occurs. In this randomized trial, we are investigating whether the use of a short feedback form, the “Jumpstart” form, can help facilitate improved communication about end-of-life care and treatment preferences. The “Jumpstart” form is based on survey questions that the patient completes at the time of enrollment into the study, and the form is shared with the patient, his/her family and clinician. The form provided to the clinician includes prompts to assist the clinician in discussing the patient’s end-of-life care and communication preferences. The effectiveness of this communication feedback intervention will be measured by comparing patients who received the “Jumpstart” form with patients who received usual care. If the “Jumpstart” intervention is effective, patients in the intervention group, as compared with those in the control group, should report improved frequency and quality of communication with their clinician and better agreement between the care they desire and the care they receive. These outcomes were measured by data in the electronic health record (EHR) and abstracted from clinic visit notes and from a series of surveys collected at three time points after the target visit, during which the intervention was implemented (2 weeks, 3 months and 6 months). If this intervention is successful, healthcare systems could implement the "Jumpstart" forms as a tool to improve patient care.

Mycobacterium tuberculosis (M.tb) is the causative agent of tuberculosis, an infectious disease responsible for nearly two million deaths around the world every year. Mycobacteria express a number of cell wall lipids that activate our immune system. For example, T cells are activated by lipids when bound to highly conserved CD1 molecules on the surface of antigen-presenting cells. T cells express a unique T cell receptor (TCR) that is the result of genetic recombination and mutation. Thus, the DNA sequence of a TCR is specific for the antigen recognized by a T cell. Virulent M.tb strains contain sulfoglycolipids (SGLs), but the diversity of SGL-specific T cells has not been determined. This information could be helpful in distinguishing infection from exposure or vaccination. The objective of this work is to determine the diversity of TCRs expressed by SGL-specific T cells. We isolated mRNA from SGL-specific T-cells that were derived from a patient with a latent M.tb infection and used template-switched PCR to amplify the TCR sequences. We will present data summarizing the diversity and conservation of sequence motifs required for recognizing SGLs. The identification of conserved sequences could be utilized to develop novel TCR sequence based diagnostics for M.tb.

Merkel cell carcinoma (MCC) is an aggressive, neuroendocrine skin cancer and one-third of patients develop metastatic disease. Specific sites and timing of distant metastases are largely un-described and, if characterized, could inform staging and surveillance protocols as well as optimize clinical  management. A cohort of 292 patients with metastatic MCC were prospectively followed and then grouped by the site(s) and timing of their initial distant metastases. Initial distant metastases were defined as the first distant metastatic lesions detected clinically and/or radiologically. Presentation of multiple lesions in a single organ site only counted as one initial distant metastasis for that organ site. MCC-specific survival was analyzed using a competing-risks regression, where 200 patients died of MCC. 292 patients had 420 distant metastatic sites, most commonly non-regional lymph nodes (40% of 292 patients), skin / body wall (26%), liver (22%), bone (20%), lung (9%), pancreas (8%), and brain (4%). Patients with only liver lesions (HR 2.1; p=0.02) or patients with lesions in multiple sites (HR 2.7; p<0.01) were over twice as likely to die of MCC than patients with distant metastases limited to the skin / body wall. Additionally, patients whose first distant metastasis occurred >3 years after initial diagnosis were twice as likely to survive MCC than patients whose MCC metastasized within two years of diagnosis (HR 0.5; p=0.02). Site of primary MCC is associated with a distinct pattern of initial distant metastasis, which may help inform surveillance practices. Survival outcomes vary among patients with metastatic MCC and prognosis may be more accurately estimated with stratification by timing and site(s) of initial distant metastasis. Accounting for these patient characteristics will help inform surveillance imaging intervals and define sub-staging for patients diagnosed with metastatic MCC.

 The percentage of obese children in the United States has tripled since the 1970’s, with one in five school-aged children now categorized as obese. Obesity development and response to intervention has been found to depend on factors such as impulsivity, though its influence as a major risk factor is unknown. This project seeks to determine if impulsivity affects weight reduction outcomes in family-based behavioral treatment. I hypothesize that increased impulsivity will be associated with increased food intake, increased food reward value and subsequently poorer weight reduction outcomes. Forty-nine obese males and females aged nine to eleven years old were evaluated before and after they participated in a 6-month family-based behavioral treatment intervention to determine the relationship between impulsivity and treatment outcomes. Impulsivity was assessed using a Developmental Neuropsychological Assessment. Each subject’s food reward value was measured using interviewer-administered tasks in which participants were questioned on their choice between preferred snack foods vs. an appealing alternative. Calorie intake was measured by providing study participants with a buffet consisting of a wide variety of foods, with which they had access to for thirty minutes. Calorie intake and composition was measured by weighing back uneaten food. Success of obesity treatment was measured in the comparison of adiposity using BMI z-scores, given that a z-score decrease of at least 0.25 improves blood pressure, lipids and insulin sensitivity. Obese children categorized as impulsive are predicted to consume more calories and rate a food reward value compared to non-impulsive obese children, resulting in poorer weight reduction outcomes, indicating that childhood obesity is correlated with impulsivity on the basis of an inability to engage cognitive control of food intake. This research will provide new insights into the neurobiological basis of child obesity, potentially improving the cost-effectively and long-term results of obesity interventions.

Effective drug delivery to solid tumors is made difficult by high cell density and poor blood flow to certain areas within the tumor. Previous studies have shown that pulsed high-intensity focused ultrasound (pHIFU), using highly focused pulses of ultrasound with a slow repetition frequency, can increase drug delivery to tissue through bubble formation. For this technology to become viable in a clinical setting, it is necessary to develop an image reconstruction technique to detect and characterize bubble activity. Passive cavitation detection (PCD) is the gold standard in the field to characterize bubble activity, but it provides minimal information on where the activity occurred. Our work focused on developing and refining previous Doppler-based imaging techniques that were shown to be sensitive to bubble formation, size and location. pHIFU pulses were delivered to optically transparent tissue-mimicking gel phantoms using either a HIFU or diagnostic ultrasound probe. The ensuing cavitation bubble activity at the focus was evaluated with three different methods: Doppler-based ultrasound imaging, PCD and direct observation using a high-speed camera. Ultrasound imaging was performed by an ultrasound probe connected to the Verasonics programmable ultrasound system, and the imaging data was post-processed in Matlab. The Doppler-based cavitation maps generated under varying pHIFU exposures and Doppler sequence parameters were then compared against PCD measurements and high-speed video recordings to select the most sensitive regime and the best quantitative metrics to characterize the cavitation-induced damage from these maps. Preliminary analysis indicates that integrated Doppler power in the region of interest correlates to the cavitation dose measured by PCD, which, in turn, was shown to correlate to the degree of tissue permeabilization.

Surveillance data has shown extremely high rates of bacterial sexually transmitted diseases (STDs) among men who have sex with men (MSM). STDs increase the risk of acquiring and transmitting HIV. HIV infection is associated with lower education levels; however, it remains unknown whether STDs are associated with lower education levels among. To determine whether education level is associated with diagnosis of bacterial STDs, we plan to conduct a cross-sectional study using baseline data from a larger cohort study on MSM and bacterial STDs. All enrollees were MSM who met inclusion criteria for either >4 sexual partners (female or male) in the past 2 months, ≥10 sexual partners in the past 12 months, or diagnosis of a bacterial STD in the past 12 months. Data consists of an enrollment demographics survey that includes age, race, educational attainment, income, number of sexual partners in the past 2 months, and STD testing results (gonorrhea and chlamydia at the pharynx and rectum, and syphilis). We will evaluate the relationship between educational level (categorized as <college education or ≥college education) and STD test results (aggregated as any positive STD result), as well as by specific STD (gonorrhea, chlamydia, and syphilis). We will also conduct a multivariable regression model to adjust for the confounders of age, race, income and number of sexual partners. Results are anticipated by April 2017. The analysis is expected to show a negative correlation between educational levels and positive STD test results. Research is needed to address the rising rates of STDs in MSM in relation to education levels.

Immunotherapy has yielded exciting results in the clinic, primarily against blood cancers. Moving toward applying immunotherapy to solid cancers, the field has seen success in treating certain cancers, such as lung cancer, with immunotherapy. However, only a fraction of patients respond to treatment, highlighting a need for continued research in this area. The ‘KP’ model is a genetically engineered mouse model of lung cancer, relying on Cre-recombinase inducible activation of oncogenic Kras and inactivation of the tumor suppressor p53, the two most frequently mutated genes in human lung adenocarcinoma. Tumors are initiated by delivery of Cre into lung alveolar cells by the administration of Cre-expressing viruses. Using this virally-induced model, we can control the timing of tumor induction and tumor burden, and introduce additional genetic modifications into tumors. However, the use of viruses can cause inflammation, potentially impacting the immune responses that we are studying. Improving this model will make the study of lung cancer more physiologically relevant. Therefore, we are developing a system for separating viral infection from tumor initiation, dependent on a tamoxifen-inducible Cre-ER allele. By developing viruses that express Cre-ER, mice can be infected and the induction of tumors can be delayed until a timepoint when virus-related inflammation has subsided. Then, Cre activity can be induced by administration of tamoxifen. Additionally, this system allows us to control the number tumors that form, by titrating the doses of tamoxifen, to better recapitulate human disease. To date, we have cloned DNA fragments and performed Gibson Assembly, and are currently validating clones. We have produced lentiviruses and titered the viruses using GreenGO cells, a cell line carrying a Green Fluorescent Protein reporter of Cre activity. To test Cre-ER functionality and sensitivity, we will use GreenGO cells to assay differences in Cre–ER activity, using different concentrations of tamoxifen in vitro.

Atherosclerosis is a disease in which cholesterol accumulates in the walls of arteries, limits blood flow, and causes heart attacks and strokes. We aim to prevent and reverse atherosclerosis with a gene therapy that increases levels of apolipoprotein A-I (apoA-I), a protein that removes cholesterol from the artery wall. This therapy increases expression of apoA-I specifically in endothelial cells (EC), cells that line the artery wall. Our initial vectors for expressing apoA-I contained the murine endothelin-1 (mET-1) promoter (an EC-specific promoter) both without (1XETE) and with (4XETE) three additional copies of an endothelin-1 enhancer (ETE). We used two approaches to increase apoA-I expression: 1) literature searches for regulatory sequences that increase EC gene expression in other contexts; 2) novel bioinformatics approaches that identify EC-specific cis-regulatory sequences (CRMs) throughout the genome based on ENCODE data, relative local abundance of EC-related transcription factor binding sites, and interspecies conservation. We ligate regulatory sequences into the apoA-I vectors mentioned above. We transfect these constructs into cultured EC and measure apoA-I expression. Using the first approach, we found that addition of a shear-stress-response element into the 4XETE vector did not alter apoA-I expression. Addition of a single MEF2C enhancer sequence into the 4XETE vector decreased expression; however, after this decline, addition of more copies of the MEF2C enhancer increased apoA-I expression. Lower expression may be due to unfavorable interactions between multiple enhancer elements. Using the second approach, we identified eleven CRMs (55-352 bp) clustered around the promoters of genes highly expressed in EC: CDH5, EFEMP1, THBS1, and VWF. In conclusion, trial-and-error addition of enhancers has not reliably improved EC apoA-I expression. A bioinformatics-based approach identified CRMs associated with high transcriptional activity in EC. We inserted the eleven CRMs into both the 1XETE and 4XETE apoA-I vectors and will test whether they increase EC apoA-I expression.

Macrophages are specialized cells of the immune system that direct the pro-inflammatory response and its resolution. Matrix Metalloproteinase 28 (MMP28) is an extracellular matrix proteinase expressed in alveolar macrophages during lung inflammation and is involved in macrophage recruitment and polarization. Our aim was to explore the regulation of Mmp28 expression in macrophages. We hypothesized that LPS (lipopolysaccharide, a component of bacterial cell membranes which simulates bacterial infection) stimulation would increase Mmp28 expression via a TLR4-dependent (Toll-Like Receptor 4) pathway involving Type I Interferon (IFN) signaling. We also hypothesized that Polyinosinic-polycytidylic acid (Poly(I:C), a synthetic form of double-stranded RNA which simulates viral infection) would increase Mmp28 by a TLR3-dependent pathway. To test these hypotheses, we generated bone-marrow derived macrophages (BMDM) from wild-type mice. We exposed these cells to purified recombinant IFNα/β (two forms of IFN involved in antiviral responses), LPS, Poly(I:C), or control media; 4-6 h after treatment, we isolated RNA from the BMDM, synthesized cDNA, and used qPCR to measure mRNA levels of several inflammatory genes. We also included BMDMs from knockout mice for several proteins thought to be part of the Mmp28 regulatory pathway. We confirmed that LPS and Poly(I:C) increased Mmp28 expression in BMDM. By comparing knockout mice for two adaptor molecules shared by TLR4, we found that LPS-stimulated Mmp28 expression was retained in MyD88-/- but decreased in TRIF-/- macrophages. Since both poly(I:C) and the TRIF-dependent response to LPS induce a type I IFN response, we tested if Mmp28 expression was dependent on type I IFN by using IFNAR-/- (IFN Receptor) macrophages. We found that both TLR4 and TLR3 ligands require IFNAR for Mmp28 expression. These data link type I IFN responses to regulation of MMP28 and development of experimental COPD and emphysema models of lung disease, and further our understanding of how MMP28 may regulate macrophage responses.

Fundamental understanding of how effector T-cells are transformed into quiescent memory T-cells is vital for developing immunotherapeutic treatments for autoimmune diseases and cancer, and for the development of vaccines against diseases such as AIDS, tuberculosis and malaria. Previous research on conversion of memory precursor effector cells (MPECs) into memory T cells has found evidence of Treg cell involvement in the process. Treg cells are regulatory helper T cells which selectively inhibit effector T cells to prevent and suppress overly strong immune responses and autoimmune responses. Research has also shown that MPECs produce their own IL-2, a cytokine involved in immune tolerance and activation. Based on these data, we hypothesize that direct interaction between MPECs and Treg cells, possibly via MPEC-produced IL-2, is required for conversion of MPECs into memory T-cells. We also addressed colocalization of Treg cells with short-lived effector cells (SLECs). SLECs are IL-2 non-producing effector cell counterparts of MPECs and do not form memory. Potential MPEC or SLEC interaction with Treg cells was visualized through immunofluorescence microscopy of the secondary lymphoid organs (spleen and lymph nodes) of experimental mice injected with lymphocytic choriomeningitis virus (LCMV). To bypass the low precursor physiological frequencies of MPECs we employed the P14 LCMV GP33-specific TCR transgenic mouse system, which is a well-regarded model system for studying memory differentiation. To test the role of IL-2 in cell-cell interactions, we used IL-2 agonist and antagonist antibodies. A detailed understanding of how Treg cells are involved in the process of MPEC to memory T cell conversion is key to developing novel immunotherapeutic treatments.

Quorum Sensing (QS) is a chemical signal based communication system by which bacteria regulate gene expression according to changes in population density. This communication mechanism is used by the environmental bacterium and opportunistic human pathogen P. aeruginosa, which is a major cause of Cystic Fibrosis (CF) lung infections. P. aeruginosa employs two interconnected QS systems: las and rhl. These systems are regulated by the transcription factors LasR and RhlR, each of which binds a unique chemical signal that further activates transcriptional regulation. LasR and RhlR together control the expression of virulence factors critical for many infection processes. In laboratory strains, the LasR and RhlR QS systems are arranged hierarchically, such that LasR transcriptionally activates RhlR. Although this QS hierarchy is common to laboratory strains of P. aeruginosa, some CF clinical isolates are capable of RhlR activation in the absence of functional LasR. However, the origin of this LasR-independent regulation remains unclear. In this study, we investigate whether the LasR–RhlR hierarchy is conserved in environmental P. aeruginosa. We collected several environmental isolates and genetically engineered them through allelic exchange mutagenesis to disrupt LasR function. We then transformed wild type and LasR-null isolates with reporter constructs that produce GFP (Green Fluorescent Protein) in response to either LasR or RhlR. In these two conditions, we measured protein levels of LasR/RhlR, signal concentrations and the production of several QS-regulated products. In most cases, we observed that RhlR activity was substantially reduced in the absence of functional LasR. The observed decrease in RhlR function is so far consistent with the idea that the QS hierarchy is a conserved feature of P. aeruginosa. However, mechanisms of quorum regulation in environmental strains may differ from laboratory strains and a proper understanding of them can potentially contribute to the development of non-antibiotic therapies.