Chronic non-bacterial osteomyelitis (CNO) is a rare disease where the immune system attacks healthy bone, leading to inflammation and destruction. Currently, the use of inflammation markers - which consists of a blood test for erythrocyte sedimentation rate (ESR) and c reactive protein (CRP), a clinical visit, and magnetic resonance imaging (MRI) are used to monitor the state of CNO. However, the reports from these assessments are inconsistent as longitudinal data allowing for a detailed analysis is lacking.Thus, we aim to investigate the association among these disease monitoring modalities using Seattle Children's Hospital CNO research database. The database which contains patients under 21 years old, spanning from January 2014 - January 2021 is one of the largest composed. We hypothesize that an increased presence of lesions, as observed through MRI, correlates with a greater value of inflammation markers and greater physician global assessment (PGA) score. Through blood samples and MRI scans, ESR/CRP values and number of active lesions within 30 days of the visit were recorded. General statistical methods were used to summarize the data and determine correlation. We expect a strong positive correlation between active lesion count and ESR/CRP values. ESR/CRP values measure the rate of inflammation. As inflammation is a common physiological symptom stemming from lesion presence, a positive correlation between these variables must occur. Additionally, we expect a strong positive correlation between active lesion count and PGA scores. PGA scores are determined during clinical visits and are drawn from assessing a patient’s current condition. The prominence/severity of lesions are factored into the PGA score. As CNO lacks a reliable technique to monitor the disease, we expect that the results from this study will shed light on a reliable and robust assessment tool to monitor disease activity.
 

Group B Streptococcus (GBS) are beta-hemolytic, gram positive bacteria that colonize the vaginal tract of 1 in 4 pregnant women, and can cause preterm birth and neonatal infections. A key factor involved in GBS pathogenesis is an enzyme known as hyaluronidase (HylB). HylB is secreted from GBS during infection, and allows GBS to evade the host immune response by interfering with pathogen associated molecular patterns (PAMPs) recognition through toll-like receptors (TLRs) 2 and 4. The goal of this project is to use a biochemical colorimetric absorbance assay to quantify HylB activity in GBS isolates by comparing 50 maternal commensal isolates to 200 neonatal invasive isolates. Following quantification, the isolates will be stratified based on the epidemiological and clinical data to determine how HylB contributes to disease outcome. Although HylB’s role in GBS pathogenesis has been partially described by in vitro experiments and animal models, this virulence factor is not assessed clinically when patients are tested for GBS during pregnancy. Thus, this project allows us to identify new correlations between HylB activity and real-world clinical outcomes, which may be important for predicting disease progression. Currently, about 24% of invasive isolates exhibit HylB activity, though analysis for these isolates are still in progress, and we are continuing this study to include commensal isolates. We hypothesize that the commensal isolates will have reduced HylB activity compared to invasive isolates, as the ability for GBS to evade the host immune response may allow for greater dissemination. Knowledge from this study may contribute to the design of novel therapeutics, such as those targeting HylB, that may help prevent poor outcomes during human infection. 

 According to the Centers for Disease Control and Prevention, birth defects affect 1 in every 33 babies born in the United States, and severity within each condition varies widely. However, the genetic basis of isolated birth defects (iBD), that is having a single major birth defect, remains largely unknown. The research question for this study is, “What are the underlining genetic causes of different iBDs?” To answer this question, I visually screened patient data obtained from the University of Washington Center for Mendelian Genomics. I looked at the DNA sequences in families to confirm the presence of a genetic variant in the affected child and identify patterns of inheritance for children affected by glaucoma or omphalocele. Glaucoma is a condition affecting vision and omphalocele is a defect of the abdominal wall that causes organs to lay outside of the stomach. I looked at these birth defects because they are common causes of infant mortality in the first year of life. Early findings show that in our cohort, 7 out of 19 individuals with congenital glaucoma have overlapping genotypes with other disorders affecting vision. In the omphalocele cohort, there were no overlaps in their genotype with other similar disorders. These results suggest that within glaucoma, there are other possible candidate genes of interest for targeted sequencing, and that these individuals may be affected by other medical conditions relating to vision. Given these findings, I will continue to analyze data for other defects to expand our understanding of the phenotypes of iBDs. The next major defect I will study is tricuspid atresia which is a defect in the heart. This research will contribute to knowledge of the genetic basis for iBDs by narrowing down the candidate genes which can be used to identify targeted sequences for DNA sequencing.

While Congenital Heart Defects (CHDs) are the most common birth defect in the US, only 20-30% of the genes that contribute to the development of CHDs have been identified. The purpose of this research is to identify the unknown genetic causes of human CHDs through a combination of Protein-Protein Interaction (PPI) network analysis and genetic manipulation of zebrafish using CRISPR. Using the ExAC human genetic database, our lab has identified over 200 new "Candidate" genes for CHDs. For this project, we sought to answer two main questions: (1) Can PPI networks serve to identify new gene interactions potentially involved in the development of CHDs? (2) Can we demonstrate functions and interactions of these new genes using mutant zebrafish embryos? To answer the first question, we utilized STRING—an online database of known and predicted protein-protein interactions—to create PPI networks that predict interactions between our “Candidate” and “Known” genes involved in human CHDs. Through PPI network analysis, we identified the interaction of five proteasome factors (POMP, PSMA6, PSMA7, PSMD3, and PSMD6). The proteasome system has been characterized to be involved in human cardiac disease, but the specific roles of these factors in heart development have not yet been determined. To address our second question, we used CRISPR-Cas9 zebrafish genome editing to preliminarily identify functions of the proteosome factors POMP, PSMA6 and PSMD6 in heart development. We then used CRISPR to create a new POMP mutant zebrafish strain. Our genotypic and phenotypic analyses confirm a critical role for POMP in heart development. This is crucial, as it demonstrates that this cluster of proteasome factors could potentially be identified as a novel grouping of genes that are related to human CHDs. Our results promise to further our understanding of the genetic causes of human CHDs.

The rhombic lip is a dorsal germinal zone that produces nearly 80% of all neurons in the human brain and assists in the production of all glutamatergic neurons in the cerebellum. In the mouse which has traditionally been used as a model organism, the rhombic lip is a transient structure and a dorsal stem cell zone that appears between embryonic days 12.5 and 17.5. While the mouse rhombic lip is molecularly compartmentalized, it lacks structural compartmentalization. The human rhombic lip on the other hand is long-lived and seen throughout gestation. It also exhibits substructure and is compartmentalized into a ventricular and subventricular zone. The extended duration of rhombic lip is a unique feature of human development and not seen in other model organisms including non-human primates like the macaque. This indicates that model organisms have limitations as they cannot fully recapitulate human development. Histological and immunohistochemical analyses of over 27 Dandy Walker Malformation (DWM) and Cerebellar Vermis Hypoplasia (CVH) samples suggest that rhombic lip disruption leads to cerebellar hypoplasia that disproportionately affects the posterior vermis. DWM and CVH are birth defects of the cerebellum that are diagnosed using ultrasound and MRI. Our histopathological analysis suggests that reduced proliferation and self-renewal of the rhombic lip progenitor pool leads to underdevelopment of the posterior cerebellum. These findings add to the understanding of the developmental pathogenesis of DWM, and underlines the need for further research of the human rhombic lip which can further improve our understanding of cerebellar brain defects.

The spatial distribution and degree of colocalization for two or more proteins, based on their fluorescence intensities, are useful metrics for phenotyping cells and informing on biological function. Yet, most commercial and open-source image analysis tools report global colocalization statistics at the image-level, and offer limited analysis at the individual cell level. To address this, we are developing a python-based image analysis pipeline to quantify robust per-cell metrics of colocalization. Our pipeline stacks image tiff files acquired on high-throughput automated fluorescence microscopes into multichannel 3D image stacks. The images are then restored via deconvolution algorithms, and cropped to remove out-of-focus image planes using Laplacian variance algorithms. For image thresholding and cell segmentation, the pipeline incorporates scripts from the Allen Institute for Cell Sciences to threshold and segment individual cells. Finally, the pipeline calculates per cell colocalization metrics based on the fluorescent intensity of each voxel in each cell. CLARITY was used to quantify the spatial relationships of the peroxisomal biogenesis protein Pex3 with the endoplasmic reticulum protein Sec61 and peroxisomal membrane protein Pmp70. Pex3 colocalized with both Sec61 and Pmp70 and this colocalization could be manipulated by treatment with different kinase inhibitors. In several instances, these differences in localization contributed to a large variance in the measured Pearson’s Correlation Coefficient of cells within the same image. Morphometric analysis showed the volume of peroxisomes per cell negatively correlated with the number of peroxisomes per cell. The automation of per-cell image analysis leveraged in this pipeline will allow for systems-level phenotyping and data mining from fluorescent microscopy images.

Mycobacterium abscessus (Mabsc) infections occur in more than 5% of patients with cystic fibrosis (CF), increasing over the last decade. Antibiotic treatment for those with the infection can take many months and often are not successful at eradicating the organism, requiring long-term maintenance therapy. Currently, there is not an effective and evidence-based drug regimen for treating Mabsc infections in patients with CF. The conventional recommended treatment consists of 3-4 drugs, which together have an eradication rate as low as 20% in Mabsc subspecies abscessus. The goals of this project were: to collect experimental data to train the INDIGO-MABSC, an in silico model for predicting synergistic and antagonistic antibiotic interactions to treat M. abscessus infections; and to identify possible synergistic interactions between antibiotic combinations. I treated liquid cultures with 40 different antibiotics for 6 hours and then isolated genomic ribonucleic acid (RNA). The RNA was then sequenced to determine gene expression changes which can be used for model construction. Using broth microdilution, I set up checkerboard assays to observe the interactions between two different drugs and assess the combination’s efficiency in inhibiting the growth of Mabsc type strain ATCC19977 qualitatively by eye. I calculated the fractional inhibitory combination for each combination. Preliminary results show putative synergistic interactions between drug pairs Cefoxitin-Rifampin, Imipenem-Cefoxitin, Imipenem-Clarithromycin, and Imipenem-Linezolid. From sequencing results, we saw that Rifampin, Imipenem, Clofazimine, Rifabutin, and Cefoxitin did not induce sufficient gene changes. Future directions include additional checkerboard testing for combinations that were inconclusive due to no minimum inhibitory concentration recorded. Additionally, I will work optimize drug treatment for antibiotics that did not induce enough gene expression changes, and also work to collect RNA from cultures treated with novel drugs and antibiotic combinations that are commercially available.

Cystic fibrosis (CF) is a genetic disorder characterized by chronic lung infections involving various organisms, including the gram-positive pathogen Staphylococcus aureus. Antibiotics, such as trimethoprim-sulfamethoxazole (SMX), play key roles in treating CF infections; these drugs inhibit bacterial growth by disrupting important bacterial metabolic processes. SMX specifically inhibits folate metabolism, causing DNA damage that results in bacterial cell death. However, S. aureus is able to persist in CF pulmonary infections despite treatment with antibiotics, and evidence suggests that S. aureus does so through adaptive mutations. Our goal is to identify the adaptive mutations of S. aureus during SMX exposure in vitro, to understand how this pathogen persists and to prevent the emergence of resistance. We grew S. aureus in the presence of super-inhibitory SMX concentrations for 24 hours in Luria Bertani broth. We sampled the culture at specific timepoints and measured viable bacterial counts on chocolate agar; we evaluated all resulting colonies for SMX susceptibility and associated genetic changes. Surprisingly, we identified mutants that survived SMX treatment carrying diverse adaptive changes not associated with folate metabolism or DNA repair, suggesting previously-unknown lethal effects of SMX against S. aureus. These mutants carried mutations predicted to decrease production of reactive oxygen species (ROS) - toxic compounds produced by all cells during aerobic respiration and in response to stress. Our results indicate ROS may play a role in SMX-mediated S. aureus cell death, suggesting that treatments that augment the effects of ROS could improve antibiotic efficacy. We are now exploring the involvement of ROS in S. aureus killing by SMX using engineered S. aureus strains with knockout and overproducing mutations in ROS detoxification genes. This study will help us better understand SMX’s mechanism of action and S. aureus’ response to this drug, in order to improve the treatment of diverse infections caused by this pathogen.

The body’s immune system utilizes T cells to identify and kill tumor cells of growing cancers. However, hematological and solid tumors inhibit T cells by producing a tumor microenvironment (TME) that presents inhibitory and death signals that hinder the T cells cancer fighting potential. At Dr. Oda’s lab we work to overcome this TME obstacle by engineering exogenous proteins that can be expressed in T cells. These immunomodulatory fusion proteins (IFPs) combine an inhibitory ectodomain with a costimulatory endodomain. Therefore, when these novel T cells receive deadly messages from tumors, they instead interpret and rewire the signal in ways that increase their cancer combatting abilities. We have had success in engineering Fas-4-1BB and CD28/CD200R IFPs data supports the IFP’s ability to increase proliferation, metabolism, and antitumor properties along with decreasing exhaustion in T cells. This presentation will focus on our recent investigations into an IFP that modulates the relationship between SIRPa and CD47. It is known that tumor cells express CD47 which binds to SIRPa on macrophages and gives an anti-phagocytic “don’t eat me” signal. CD47 also binds to SIRPa on dendritic cells and shuts them down, effectively inhibiting an antigen presenting cell that is key in communicating cancer antigens to T cells for identification. By combining the SIRPa ectodomain with the costimulatory CD28 endodomain, we can manipulate this inhibition by CD47 which is overexpressed in TMEs. I use gateway cloning to prepare vectors for transfection. I transfect SIRPa/CD28 constructs into PlatE viral producing cells and transduce the virus into T cells for expression. From here, I set up serial killing over time assays to collect and analyze in vitro data. This research on IFPs can lead to improved clinical treatment for cancer patients, resulting in increased survival rates across a range of different cancers and patients of all ages.

Our immune system naturally fights cancer by using T cells to locate and infiltrate cancer cells. However, hematological and solid tumors counter this by presenting inhibitory and apoptosis-inducing ligands on their surfaces, effectively suppressing T cell activity. We address this barrier in cancer treatment with studies on genetically engineering T cells. Specifically, we are engineering T cells with a Fas-4-1-BB immunomodulatory fusion protein to overcome inhibitory signals and instead utilize them to enhance the T cells’ cancer-combatting abilities. Inhibitory signaling from Fas ligand (FasL) plays a major role in the proliferation and persistence of tumors in the body by protecting tumor cells from being attacked by lymphocytes. We are engineering T cells with an immunomodulatory fusion protein (IFP) that combines the Fas ectodomain on T cells with the pro-survival 4-1-BB signaling domain. This allows T cells to convert the inhibitory Fas signal from binding FasL that diminishes T cell activity to costimulatory 4-1-BB signal which upregulates T cell activation and survival. What we found is T cells transduced with the Fas-4-1BB IFP exhibit enhanced ability to proliferate and function in vitro. Fas-4-1BB T cells also exhibit metabolic changes that lead to increased spare respiratory capacity (SRC) and mitochondrial biogenesis. KPC pancreatic cancer and FBL leukemia models in mice show improved persistence and survival in T cells expressing the Fas-4-1BB IFP. In conclusion, our approach in T cell engineering with IFP allows for T cells to overcome deadly signals from tumors and interpret them as a pro-survival message, thus increasing T cell function in murine models. Our results suggest that such engineered T cells improves efficacy towards fighting both solid and hematological tumors.

Chronic nonbacterial osteomyelitis (CNO) is an autoinflammatory bone disease associated with persistent bone pain, destruction and pathological fractures. While physical findings and laboratory tests are used in monitoring CNO disease activity, these methods are not yet reliable. Laboratory tests for N-terminal telopeptide (NTx) in the urine and C-terminal telopeptide (CTx) in the serum can be used to measure bone resorption that results from the breakdown of affected bones in CNO. These tests can serve as a useful monitoring marker in CNO patients taking bisphosphonates because these medications work by inhibiting osteoclasts that cause the degradation of bone. We aimed to assess the dynamic change of urinary NTx and serum CTx in children with CNO during and after treatment with bisphosphonates to determine if there is a correlation with disease activity. After IRB approval, we obtained consent from participating patients and families . Blood and urine samples, MRI scans, and clinical data including Physician Global Assessment (PGA) were collected during clinical visits at Seattle Children’s Hospital. We analyzed collected data using descriptive statistics and performed a correlative analysis. Based on our small cohort, we confirmed an initial decrease of NTx and CTx during bisphosphonate treatment in patients with improved PGA scores. After treatment, disease flare among these patients was not clearly correlated with the subsequent increase of these two markers; however, a well-controlled large-scale study is warranted for further confirmation. Results of this study have the potential to improve the efficiency of disease monitoring methods for pediatric CNO.

Vulnerable immigration statuses in Latinx children in the US are often associated with poor health, development and social service access. This adversity can be mitigated by positive and responsive parenting that promotes resilience and supports the childhood stress of migration. In this study, we aimed to understand the parenting strengths, stressors and law needs of Latinx families in order to adapt a brief, responsive parenting behavioral skills intervention—Social, Emotional, and Academic Competence for Children and Parents (SEACAP) and pair it with immigration law and policy education and resources. We conducted 15 qualitative interviews, 30-60 minutes in length, with Latinx parents of young children with vulnerable immigration status. The interviews were conducted by a bilingual-bicultural member of the study team in Spanish. The interviews were recorded, transcribed and organized using Dedoose software; and reviewed by myself and additional research associates using thematic analysis coding methodology to segment and categorize important concepts within our dataset by generating codes. A codebook was created with the consensus of a coding team using Dedoose software. There have been a total of 15 qualitative interviews conducted, transcribed, coded and are currently in process of further thematic analysis. Preliminary major code themes that have been revealed include: Migration Strengths and Stressors, Stress Level Comparison, Push/ Pull factors and Health. The themes identified in the ongoing analyses will inform the adaptation of the SEACAP curriculum and the development of the law and policy education and resources to support Latinx families with vulnerable immigration status. Implemented through community partnered organizations, this program aims to provide parents with information about warm, consistent parenting and scaffolding to improve children’s development of self-regulation and positive social, emotional and academic outcomes. This program will also include legal advice and information related to immigration and education, offered by the North West Justice Project.