We are investigating adverse event (AE) reporting in clinical trials on cystic fibrosis (CF) to address whether specific approaches to coding AEs prior to un-blinding of study data is informative. While attribution standards are available in the Common Toxicity Criteria; there are no formal guidelines on how this determination should be made. The process of reporting attributions increases both time and cost of the trial although it is uncertain whether this process is accurate or valuable. This is a retrospective data repeated measures data analysis with pooled clinical trial data from four randomized controlled trials involving patients with CF. We will employ descriptive statistics to describe the overall population and demographics from individual studies who experienced at least one adverse event and summarize the adverse event rates over all and per study by individual rates. We will also employ repeated multiple variable regression techniques to describe associations of demographic and clinical characteristics with the attribution of AE’s. We hypothesize that physicians are not very good at attributing these events when the patient is on the placebo. We currently have available data on 11,974 adverse events in 785 subjects in the four randomized controlled trials. This study could have implications to how clinical trials are conducted in the United States. A future goal would be to clarify the value of attribution of adverse events in clinical trials.

Blood clotting after injury requires processing of Von Willebrand Factor (VWF), a large blood protein that self-associates into long fibers which are then broken down by the enzyme ADAMTS13 into smaller fragments. These cleavage fragments self-associate after injury to stabilize clotting factors and activate platelets for clot formation. When the activity of ADAMTS13 is insufficient or inhibited, overactive clot formation leads to thrombotic thrombocytopenic purpura (TTP). VWF fiber formation can be modeled using a microfluidic device that measures the flow of platelet-free plasma through a microscopic channel. My goal is to investigate the properties of cleaved VWF fragments and how they regulate the capacity of ADAMTS13 to further cleave normal VWF. I hypothesize that the presence of cleaved VWF by ADAMTS13 will inhibit further cleavage and produce longer VWF fibers. To test this hypothesis, I will examine VWF self-association in the microfluidic channel - in the presence and absence of the cleaved VWF fragments. The addition of VWF fragments will be expected to inhibit further cleavage of VWF and increase self-association through the microfluidic channel. Understanding how cleavage products from ADAMTS13 breakdown of VWF can regulate self-association of VWF will provide critical insight into causes of TTP and developing novel treatments for patients with this disease.

Merkel cell carcinoma (MCC) is an aggressive skin cancer with a propensity for early dissemination. As little is known about this process, we sought to identify the preferential sites of initial distant metastatic MCC in order to guide imaging evaluation and clinical follow up. In this retrospective study, we analyzed the clinical and imaging records of 442 initial distant metastases from 305 MCC patients enrolled in our data and tissue repository. Initial distant metastases were defined as the first lesions detected beyond the regional lymph nodes of the primary tumor. Among 305 patients, 69% had 1 initial metastasis, 19% had 2, 9% had 3, and 3% had 4 or more. Of the 305 patients, 50% were evaluated only by radiologic and/or clinical criteria, while 50% also underwent biopsy-confirmation of MCC metastases. Of the 442 initial distant metastases, the distribution of sites was: 26% to distant lymph nodes (typically supraclavicular, retroperitoneal, or iliac), 15% to liver, 13% to skin, 13% to bone, 13% to other visceral sites, 6% to lung, 5% to pancreas, 3% to brain, 2% to kidney/adrenal gland, 2% to gonads, 1% to chest wall, and 1% to stomach. Although there were similarities in initial metastatic sites between MCC and melanoma, the brain (3%) and lung (6%) were markedly less common in MCC than in melanoma (20% and 36%, respectively), while the pancreas was more common in MCC (5%) than melanoma (<1%). Given the aggressive nature of MCC, with ~40% of patients eventually developing distant metastatic disease, these findings may facilitate improved detection and management.

To support the increased production of hemoglobin, the oxygen-binding protein in red blood cells, erythroid progenitor cells need large amounts of its functional constituent biochemical, heme. Despite this increased demand for heme, excessive amounts of free (unutilized) heme form reactive oxygen species which causes damage to cellular components and even apoptosis. To understand the decisions individual cells make to compensate for induced excessive heme, we are analyzing expression data from a pilot single cell RNA-sequencing study. Alongside various exploratory revelations, we identified Tspo (Translocator Protein), a poorly characterized mitochondrial transmembrane protein as one with significant expression changes in cells with heme excess. I have been conducting bioinformatics analyses on the transcriptomic data to classify it's expression pattern and find possible correlations with the expression of other genes. As per my initial findings, it's expression resembles that of a ROS-response gene, which will guide my further molecular/ biochemical experiments into determining causation vs. correlation between Tspo expression and heme excess. I intend to measure TSPO expression alongwith several other genes implicated in pathways related to it under conditions of upregulated heme synthesis. I will also quantify expression patterns through erythroid development in vivo with induced heme overload. I hope to better characterize the role of Tspo, if any, in heme-response pathways with these experimental investigations, together with bioinformatics analysis on large-scale transcriptomic data.

Systemic immune activation (IA) is a proven key factor in human immunodeficiency virus type 1 (HIV) infection pathogenesis and progression to acquired immunodeficiency syndrome. The reproductive hormone progesterone plays a role in regulating the immune system, and most cells contain progesterone receptors. Previous studies have linked high-progestogen states in women, such as pregnancy and progestin-based contraception use, to increased infectivity in HIV-seropositive women and increased HIV acquisition in seronegative women. Progestin-based contraceptives such as depot-medroxyprogesterone acetate (DMPA) can create high plasma levels of progesterone analogs. Therefore, studying DMPA use by HIV-seropositive and seronegative women may clarify DMPA’s role in HIV-related IA and genital shedding, and immune changes related to HIV acquisition. We hypothesize that DMPA increases systemic IA. Increased IA could be clinically significant by contributing to increased HIV acquisition in high-risk HIV-seronegative women, and by increasing genital shedding and infectivity among HIV-seropositive women. To answer this question, peripheral blood mononuclear cells (PBMC) were collected at three postpartum time points over six months from 120 Kenyan women with both HIV-seropositive and seronegative status, including a sub-population in each category that elected to receive DMPA for contraception. Two immunophenotyping panels were developed to measure T-cell IA and cellular subsets of interest using flow cytometry (CD38, HLA-DR, Ki-67, Bcl-2 for T-cell activation; FOXP3, CD25, CD127 for regulatory T-cells; CCR7, CD45RA, CD27, CD28, CD57 for memory subsets; CCR5 for the HIV co-receptor). With preliminary analysis completed for most samples, specific antibody populations have been gated with FlowJo software. After completion, we will compare different population percentages in the four arms of the study, observing how these change with DMPA administration. These calculations will help elucidate expected differences in inflammatory and regulatory changes in both HIV-seropositive and seronegative women with respect to the relationships between DMPA, immune activation, and HIV susceptibility and infectivity.

Epigenetic analysis is a rapidly growing field of interest within the research community, due to its potential to advance our knowledge of the origins of a variety of diseases. It involves analyzing modifications of DNA as well as modifications of proteins associated with DNA, which affect overall chromatin structure and lead to potentially detrimental effects on gene expression. Aberrant gene expression has been shown to be involved in the development of diseases such as cancer, diabetes, and kidney disease. The most commonly used method for epigenetic analysis is chromatin immunoprecipitation (ChIP). However, current ChIP assays involve time-consuming and labor-intensive pre-analytical sample preparation and have low throughput, resulting in concerns about accuracy and sample loss. Thus, the current assays are limited in their clinical utility. The purpose of this project is to design an optimized protocol for DNA-protein crosslinking and chromatin shearing using novel high-throughput ultrasound technology for increased efficiency and reduced sample loss in ChIP. Various conditions were tested with different stages of ChIP, including DNA-protein cross-linking, and chromatin shearing. Effects of these conditions on the ChIP assay were analyzed utilizing a microplate-based matrix ChIP assay and quantitative polymerase chain reaction (qPCR). Efficacy of chromatin shearing was analyzed using gel electrophoresis. My findings will contribute to the development of a fully automated platform for chromatin shearing and ChIP, and lead to advances in epigenetic biomarker research of diseases that burden our society. Its application to human tissues will allow for quick and accurate evaluation of chromatin alterations and gene-bound enzymes, factors, and receptors in a wide variety of human biospecimens. It will also allow for discovery of innovative diagnostic and therapeutic biomarkers based on epigenetic pathways, and determination of genomic pathways that are associated with drug resistance development.

 1 in 600 people world-wide are diagnosed with polycystic kidney disease (PKD), which leads to reduced kidney function over time. A cure for PKD is not currently available, making it important that we find more effective therapies for this disorder. Our laboratory has successfully used pluripotent stem cells to generate kidney organoids that functionally model the physiology of the kidney. The goal of this study is to generate induced-pluripotent stem (iPS) cells, adult somatic cells which have been reprogrammed to an undifferentiated state, from individuals affected with PKD. The first step of the process is the isolation of the patient’s cells from hair and urine samples. We are then able to reprogram these disease-affected cells into iPS cells and later differentiate them into kidney cells to create a better model for the disorder and screen for novel drugs. The information we learn from this study will help us better understand how PKD progresses and improve its treatment. We also hope that one day we will be able to transplant healthy kidney cells back into their patients, eventually eradicating the need for kidney transplants.

While there are currently drugs available to treat patients with human immunodeficiency virus (HIV), there is no known cure for this infection. As the HIV virus integrates into the host’s DNA, reservoirs of latent HIV are established. Because antiretroviral drugs only attack replicating HIV, the presence of these latent reservoirs prevents the eradication of the virus from the body. Once the use of anti-retroviral drugs is discontinued, the active virus reemerges in patients. The purpose of this project is to identify transcriptional and epigenetic pathways to reverse HIV latency, knowledge that could pave the way for new drug design to kill all existing virus in the body. To accomplish this goal, we are using a microplate-based matrix chromatin immunoprecipitation (ChIP) assay and quantitative polymerase chain reaction (qPCR) to measure more than one hundred different transcription and epigenetic events along the latent HIV genome upon its transcriptional activation. We applied cluster analysis to our data and have found that Protein Kinase A (PKA), a class of enzymes which phosphorylate proteins, and its target, the transcriptional factor CREB, are recruited to HIV genome. This finding, which has not previously been reported, suggests that PKA plays a role in HIV genome transcription, and if so, could be a possible path for developing pharmacological reversal of HIV latency.

More than 20 million Americans have some level of chronic kidney disease. With the constant deterioration of kidney function, treatments for this disease are in high demand. The only treatments available today include dialysis and kidney replacement. Unfortunately, these methods are not ideal due to a high dependency on the dialysis machine, long waiting lists, and possible rejection of the organ. Recently, protocols were developed that allow us to use pluripotent stem cells to generate kidney-like organoids. These organoids contain major structures of the nephron, the functional units of the kidney, which clean and filter the blood. For instance, podocytes, proximal tubules, distal tubules, and various cell types develop in these samples. This complexity that develops allows more accurate modeling of kidney disease than previous methods. A new protocol developed by our lab allows us to grow miniature organoids in microwells; this will allow us to perform large scale drug screens to search for treatments for various types of kidney disease. The miniature organoids were tested to see if they replicate the complexity seen in larger wells. Immunofluorescence was used to characterize the kidney organoids and the different cell types present within them. Additionally, proteins with the capability to perform biological functions, such as transporting glucose and other ions, are able to be studied as well. The results were compared to human fetal kidney tissue. The miniature organoids replicate all of the same structures found in the larger wells. Because of the strong similarities between miniature kidney organoids and the human kidney, they are optimal for drug testing. Impacts associated with new therapies will be easier to monitor through the miniature kidney models.

Macrophages play a large part in the immune response to Mycobaterium tuberculosis (M.tb) infection, and its response to infection is modulated by pattern recognition receptors. CD43 is a receptor glycoprotein on the macrophagal surface that has been shown to be associated with differential response severity of TB. The mechanism behind CD43 involvement in the macrophage response to M.tb is still unknown. Our hypothesis is that CD43 controls M.tb growth in human macrophages by mediating pro-inflammatory modulators. We knocked out the CD43 gene in the monocytic cell line THP-1 using the gene editing system lentiviral CRISPR Cas9. Using this cell line, we investigated how the knocked-out cells were affected when infected with M.tb compared to wild-types. We did this by measuring replication of a luminescent M.tb strain. Additionally, since M.tb capsule protein CPN 60.2 is a known ligand of CD43, we used CPN 60.2 to stimulate a tumor necrosis factor (TNF) cytokine response in both wild-type and knocked-out cells. We evaluated the TNF response using an ELISA assay. We found a statistically significant higher rate of replication in the CD43 knocked-out cells compared to wild type cells, indicating that CD43 plays a significant role in the macrophage’s pathway of killing M.tb. We also found significantly lower TNF secretion in CD43 knocked-out cells when compared to wild type cells after stimulation with CPN 60.2, which shows that cytokine response to M.tb is likely modulated by CD43 as well. Taken together, these results show that CD43 is a novel receptor on the macrophage surface that modulates the innate immune response to M.tb by controlling growth and pro-inflammatory cytokines such as TNF. The next steps in this project are to explore the exact mechanisms, such as M.tb macrophagal uptake and phagosome formation, that CD43 targets to control M.tb growth to develop targeted therapies for better TB treatment.

Acute myeloid leukemia (AML) is characterized by uncontrolled proliferation of abnormal immature bone marrow progenitor cells (“blasts”). AML blasts reside in the bone marrow microenvironment via adhesion molecules such as CXCR-4 and VLA-4, which confer protection from chemotherapy in AML. L-selectin is a surface receptor utilized by leukocytes (white blood cells) for rolling on the endothelial lining of blood vessels and adhesion to sites of inflammation. CXCR-4 is a chemokine receptor for stromal derived factor (SDF-1, CXCL12) produced by bone marrow stromal cells. VLA4 is an α4β1 integrin that mediates adhesion to alternatively spliced fibronectin and cellular vascular cell adhesion molecule 1 (VCAM1). Previous studies in our laboratory and others suggested that expression of CXCR-4 and VLA-4 is associated with prognosis in AML, and L-selectin expression increases with older age in AML patients. The hypothesis of my project is that L-selectin expression level is associated with chemotherapy resistance in AML. We performed experiments on blasts with high vs. low L-selectin surface expression as measured by flow cytometry assessment of mean fluorescence intensity (MFI). We allowed the AML blasts to be attached to plates coated with PSGL-1, an L-selectin ligand, or bovine serum albumin (BSA), a control protein. Then blasts were treated with cytarabine (Ara-C), one of the most active chemotherapy drugs in AML, for 72 hours. Data were collected by manual counting of viable cells, Cell-Titer Glo luminescent viability assay, and flow cytometry for Annexin V, as a measure of apoptosis. Our data showed that PSGL-1 binding protected AML blasts from chemotherapy in 4/15 (27%) of the samples, which had higher L-selectin (p = 0.05), lower VLA-4 expression (p = 0.10) and higher CXCR-4 (p=0.11) surface expression. Therefore, these results suggest that differences in expression and function of L-selectin, CXCR-4 and VLA-4 are associated with chemotherapy sensitivity in AML blasts.

Human immunodeficiency virus (HIV) infection is an important health issue worldwide and consists of two main types, HIV-1 and HIV-2. In the fight against HIV, integrase strand transfer inhibitors (INSTIs) are a relatively newer class of antiretroviral drugs aimed at blocking viral replication. FDA-approved first generation INSTIs include raltegravir (RAL) and elvitegravir while the second generation includes dolutegravir and cabotegravir (CTG). The high mutation rate of HIV often results in resistance to antiretrovirals and leads to treatment failure. However, the mechanisms and mutations that confer INSTI resistance in HIV-2 are not completely understood, despite the growing importance of INSTIs for HIV-2 treatment. The purpose of my research is to identify and characterize genetic changes responsible for INSTI resistance in HIV-2. Specifically, I engineered mutations that have been reported in sequences from patients receiving INSTI treatment into a laboratory strain of HIV-2, and tested the mutants for resistance to RAL and CTG in culture. Initial results indicate that a mutation at integrase position 263 (R263K) does not confer resistance to either RAL or CTG. However, a second variant encoding the mutations G140S+Q148H+N155H is highly resistant to both drugs, with 50% effective concentrations (EC₅₀ values) that were at least 1000-times greater than the wild-type strain. Experiments with additional HIV-2 integrase mutants are in progress. In the future, the mutant HIV-2 clones produced in this study will serve as a reference library for evaluating the resistance profiles of newer integrase inhibitors, thus helping to identify therapeutic strategies for HIV-2 patients that harbor drug-resistant virus.

Cardiovascular disease is the leading cause of death worldwide due largely to the inability of the heart to respond effectively to injury through tissue regeneration. Coronary artery narrowing initiates cessation of arterial blood supply to the heart generating an ischemic injury, characterized by low oxygen and nutrient supply and the accumulation of metabolic waste. Currently, research approaches to study ischemia rely on high-cost and low-throughput in vivo models. As such, there is a need for an in vitro platform for mimicking ischemia/reperfusion injury in a three dimensional cellular environment. The research and design pursued here addresses this deficit in modeling capabilities by developing an in vitro ischemic injury and reperfusion model utilizing engineered cardiac micro tissues. Cardiomyocyte microtissue particles (MTPs) are formed through centrifugation of a single cell suspension of stem cell derived cardiomyocytes without scaffold materials into polydimethylsiloxane micro-wells. Ischemic injury is induced in MTPs by exposing tissue cultures to mineral oil layers on top of small volumes of spent culture media to restrict oxygen supply and metabolic waste exchange. This simulated injury results in an observable reduction in live cells as evidenced by a decreased live cell fluorescence signal associated with duration of injury from a calcein and EthD-1 live/dead stain. Optimization of this staining protocol for characterizing the extent of the induced injury in an adapted plate reader format is currently underway. Thus far, percent of live cells has been shown to positively correlate with normalized calcein fluorescence intensity and current efforts are directed towards increased efficacy of EthD-1 dead cell marking and sufficient tissue infiltration of the stain. Implementation of this protocol will create a baseline metric for monitoring the state of the engineered cardiac tissue during injury and reperfusion and provide a foundation for development of an accurate and cost effective cardiac ischemia injury model.

Cytomegalovirus (CMV) infection is a common chronic viral infection that can cause significant complications in bone marrow transplant (BMT) recipients and other immunocompromised people. Using stored serum from BMT patients who were CMV negative but had a CMV positive donor, we have identified a group of patients who appear to have had self-limited infection, where DNA from CMV was found in their serum then resolved without therapy. It is thought that donor-derived, entry specific neutralizing antibodies (NAbs) bind to the virus and prevent entry into cells and infection. In this study, we measured the CMV NAb activity in patients with self-limited infection and compared this to similar patients who developed chronic infection. Specifically, serially diluted patient sera was incubated with a standard quantity of CMV, and the relative decrease of cellular infection was assessed. Our CMV strain contains a green fluorescent protein (GFP) gene that is expressed by infected cells. The virus-serum mixture was incubated with human epithelial cells for one hour, and then washed away. On day 7, we measured the fluorescence emitted by infected cells and constructed a standard curve to compare the fluorescence of the cells with sera to those without virus and those without serum. We calculated the serum concentration required to neutralize 50% of the virus (IC-50). Because NAbs prevent CMV entry into cells, those with self-limited infection may have greater NAb activity (i.e. IC-50) than patients who develop infection. We have measured the IC-50 in serum from 7 healthy donors. These preliminary data indicated that people with high NAb activity have a log₂IC-50 of 9-11, where CMV negative people have a log₂IC-50 <3. We are now measuring the NAb activity of patient samples. If NAb activity is important for prevention of infection, this may aid development of CMV vaccines and therapeutics.

Cytomegalovirus (CMV) is a common herpes virus that causes severe illness in immunocompromised patients, such as transplant recipients. Due to scarcity of matching donors, it is sometimes necessary to give a patient who is CMV-negative hematopoietic stem cells from a donor who is positive, resulting in a 25% risk of infection. A study was performed 30 years ago to determine if giving patients immune globulin with high levels of CMV-specific antibodies (CMV-IVIG) could prevent infection. The hypothesis was that CMV-specific antibodies would bind to and neutralize the virus before it could establish infection in the patient. The results demonstrated decreased risk of infection among patients who received CMV-IVIG compared to those who did not (controls). However, because of the poor diagnostic and treatment options available for CMV at this time, there was no difference in the rate of illness or survival, so the strategy was abandoned. Using samples from this trial, we will measure the ability of antibodies to prevent CMV entry into epithelial cells utilizing a novel neutralizing-antibody (NAb) assay. A standard concentration of a green fluorescent protein (GFP) labeled, CMV strain is added to dilutions of patient sera. The virus and sera solutions are overlaid onto epithelial cells for one hour and then washed. The cells are incubated for one week and the intensity of GFP fluorescence is measured. We construct a standard curve and extrapolate the concentration of serum required to neutralize 50% of the virus (IC-50). We hypothesize that patients who received CMV-IVIG will have higher NAb activity than controls. We have completed testing of 17/63 patients. The mean log₂IC-50 of patients who received CMV-IVIG (n=8) was 6.25, compared to 5.33 in controls (n=9). Identification of a marker that predicts prevention of CMV infection would be important for CMV vaccine development.

Recent proposed guidelines for Alzheimer’s disease (AD) stress the importance of AD subtypes (DuBois et al., Lancet Neurology 2014). There are limited data available regarding incidence rates of AD- and non-AD dementia subtypes. Better understanding of these subtypes is an important objective for improving clinical diagnoses and planning future research targets. Using data from the Adult Changes in Thought (ACT) Study we identified groups of individuals with pronounced deficits in memory, executive functioning, language, visuospatial ability, and attention, defined as >1SD below their average score on the battery of cognitive tests administered at the dementia evaluation. We performed two sets of analyses on these groups. The first set of analyses was on the subset of the ACT cohort who died and came to autopsy (636 individuals). In this group, we analyzed Braak stage and CERAD level, which are measures of disease progression, using data from 274 cases and 344 controls, as well as the presence and location of microinfarcts, macroscopic infarcts, Lewy bodies, and amyloid angiopathy. The second set of analyses was on the entire ACT cohort regardless of autopsy status. In this group we analyzed dementia onset, years of education, self-reported race, and medical comorbidity as estimated from pharmacy data. We also analyzed APOE genotype in this group; one variant of this gene is a well-know risk factor for AD. In ACT, of 867 cases of dementia with probable or possible AD, 745 (86%) had complete data with non-missing scores for all cognitive domains. Of these, 517 (69%) had a typical cognitive profile while 228 (31%) had an atypical cognitive profile. We concluded that clinical AD with a typical pattern of cognitive deficits is the most common form of incident dementia in the community, and that clinical AD with an atypical pattern of cognitive deficits is more common than non-AD dementia. Further work may reveal associated risk factors for these groups.

Chlamydia trachomatis is an obligate, intracellular, Gram-negative bacterium that infects approximately 106 million people annually, making it the most common cause of bacterial sexually transmitted infection worldwide. These staggering statistics reflect the substantial lack of scientific understanding regarding the pathogen’s developmental cycle, creating a huge obstacle for effective vaccine development. So far, it is known that after binding and host cell entry, Chlamydia replicates within a vacuole called an inclusion. Once replication is complete, exit occurs by two different pathways, one of which is the lysis pathway. This pathway specifically involves the release of free, infectious bacteria by inclusion and host cell rupture. Results from previous experiments strongly indicate that cysteine proteases serve a crucial role in inclusion membrane degradation central to the lysis pathway late in the developmental cycle (about 48 hours post-infection). My primary research goal is to apply a novel labeling approach called activity-based protein profiling (ABPP) to determine the identity of the specific chlamydial cysteine proteases involved as a critical first step towards determining the molecular mechanisms of the chlamydial lysis exit strategy. ABPP utilizes a specific probe consisting of a reactive group, which covalently binds to the active site of the protease, and a tag, which is usually a fluorophore or affinity label. After extensive optimization of probe concentrations, I exploited this beneficial specificity to isolate successfully labeled cysteine proteases from Chlamydia-infected cells and assess proper probe binding via SDS-PAGE gels. Following isolation and purification, samples will be sent to collaborator Dr. Aaron Wright at Pacific Northwest National Laboratory for mass spectrometry analysis. Successful identification of these proteases will confirm which specific proteases are essential to lysis, enhancing our understanding the Chlamydia lysis pathway and allowing for evaluation of future vaccine targets to reduce the global burden of Chlamydia transmission.
 

Besnoitia besnoiti is an emerging apicomplexan parasite that causes bovine besnoitiosis. Found originally in Africa, and now in many European countries. It causes, among many other things, decreased milk production, sterility in bulls, and an increased mortality rate, which all have severe economic affects. Currently, there is no cure, no publicly available genome sequence, and only a very unreliable vaccine. Our goal is to develop a new drug for the treatment of bovine besnoitiosis. To do this, we will find compounds that inhibit the enzymes that are crucial for the survival of B. besnoiti. Calcium dependant protein kinases (CDPKs) have shown to be promising targets in other closely related enzymes. They are essential for the survival of the parasite and can be targeted by bumped kinase inhibitors (BKIs) which bind to the ATP binding site of these enzymes. We have identified, cloned, expressed, and assayed recombinant BbCDPK1 for the inhibition of its enzyme activity with 63 BKI analogs using previously described luciferase assay. BbCDPK1 was inhibited by low concentrations of BKIs. Experiments so far have had around a 90% inhibition rate, and four compounds in particular have been very effective with only small amounts of compound. Further experiments are planned in vivo, as the BKIs have shown no toxicity in cattle. Our studies thus far indicate that this strategy could lead to non-toxic, selective CDPK1 inhibitors with good PK properties, and are thus excellent leads for further besnoitiosis drug development programs.

A Mycobacterium bovis-based Bacillus Calmette-Guerin (BCG) vaccine is protective against childhood TB meningitis but insufficient for controlling the disease. The development of efficacious vaccines requires mechanistic understanding of host immune response to MTb and the BCG vaccine. We have previously identified that single nucleotide polymorphisms in the gene HSP90B1 are associated with development of TB disease in South African children. HSP90B1 is a molecular chaperone for integrins and most Toll-like receptors (TLRs). TLRs recognize pathogen-associated molecular patterns (PAMPs), and are critical for the innate immune response to infection. In a mouse model, HSP90B1-deficient macrophages were unresponsive to ligands that target TLR2, 4, 5, 7, and 9. Our lab has generated an HSP90B1 gene knockout using CRISPR/Cas9 technology in a human monocyte cell line, and I have confirmed successful mutagenesis via a restriction fragment length polymorphism analysis. Our preliminary data indicates that exposure to TLR ligands PAM2, PAM3 and LPS, in addition to MTb whole cell lysate is correlated with a reduction in Tumor Necrosis Factor (TNF-α) secretion in the knockout cells compared to controls. We hypothesize that HSP90B1 is a critical modulator of the MTb-induced TLR signaling pathway, which drives the production of inflammatory cytokines and plays a key role in the immune response to MTb. My objective is to characterize the effects of HSP90B1 on the TLR signaling pathway by stimulating cells with TLR ligands such as flagellin for TLR5, imiquimod for TLR7, and performing cytokine quantification via enzyme-linked immunosorbent assays (ELISA). Moreover, I will conduct Griess assays to evaluate effects of HSP90B1 upon nitric oxide production, which is an important defense molecule in enhancing efferocytosis, the process of up taking apoptotic cells. These studies will improve our understanding of host immune responses to MTb and potentially lead to development of more efficacious TB vaccines.

 Each year about 33 children 18 years of age or younger are hospitalized and 19 die from firearm injuries in Washington State. Safe firearm storage, including storing firearms locked, unloaded, and separate from locked ammunition is associated with a lower risk of such injuries. The purpose of this study is to evaluate the effectiveness of 2 community-based firearm safety events that included the provision of a free firearm locking device (lock box or trigger lock) among firearm owners in Washington State, particularly among families with young children in the home. At each event, participants received training to operate the safety device and advice to store their firearms safely. Participants were asked to complete voluntary onsite questionnaires that established household firearm storage practices prior to receiving the lock box or trigger lock and consent to follow-up phone surveys approximately 4-6 weeks later. Surveys included questions assessing demographics, motivations for firearm storage practices, level of comfort with potential firearm safety counselors (e.g., physicians, law enforcement officers), and changes in household firearm storage practices since the safety event. Participants were compensated for their time with $5 gift certificates. Of 398 event participants, 195 completed both surveys and were included in the study sample. About one-half were female and 81.4% reported keeping a firearm in the home. The results from this research will help determine the best way to improve gun safety within the firearm community.

One limitation of chemotherapy drugs is their impaired ability to penetrate tight junctions between epithelial cancer cells to access the inside of a tumor. Adenoviruses have devised a method for moving between epithelial cells by virtue of binding adenoviral fiber proteins to the human protein desmoglein-2 (DSG-2) found within these junctions. When used in coordination with chemotherapy drugs, a recombinant adenoviral fiber protein, termed JO-1, was shown to improve the efficacy of the cancer drug by opening tight junctions thereby facilitating entry of the drug into the tumor mass. Mutagenic library studies with JO-1 identified point mutations that resulted in increased affinities for DSG-2. An affinity enhanced variant, JO-4, containing a V239D mutation is currently being evaluated for tumor co-therapy. Two additional mutations were identified, one (N293S) lead to an increased association rate, and a second mutation (Y250F) slowed dissociation by stabilizing the complex between the fiber protein and DSG-2. We hypothesized that combining multiple advantageous mutations in a single protein would result in an overall additive effect on affinity and activity. Utilizing more active proteins in cancer therapy can allow for smaller doses and higher potency of chemotherapy drugs in human patients. Two proteins based on JO-4 and containing the Y250F or the N293S mutations were designed in silico, cloned, and produced in E. coli. These proteins were then characterized for functional activities based on the ability to compete with adenovirus infection and the ability to enhance chemotherapy in mouse tumor models.  We expect the proteins with heightened affinity to DSG-2 will block virus infection at lower doses than JO-4 and in conjuction with chemotherapy drugs will stunt the growth of tumors in mice compared to chemotherapy alone.