Cannabis is one of the most commonly used recreational drugs in the United States. Its effects are mediated by cell surface receptors binding to cannabinoids found in cannabis, and expression varies with location and cell type. Endocannabinoid receptor 1 (CB1) expression occurs primarily in the nervous system while endocannabinoid receptor 2 (CB2) expression is commonly associated with immune cells. Cannabinoids can affect the immune response, however their specific effects on the immune response to pulmonary viral infection is unknown. In these experiments we use a mouse model to study the effects of cannabis smoke inhalation on subsequent influenza virus infection. C57BL/6 wild type mice or CB2 knock-out mice were exposed to a sub-acute level of smoke for 2 to 4 weeks using a TE Manual Smoking Machine. Cages of mice were exposed every other day to two rounds of smoke from six cannabis cigarettes. Each smoking round lasted 30-60 minutes with a 10-15 minute recovery period. Tetrahydrocannabinol (THC) levels, an active component in cannabis, were assayed by mass spectrometry in urine and blood. THC levels (0.74 +/- 0.17 ng/mL in urine, 14.3 +/- 0.94 ng/mL in blood) indicate an effective smoking regimen. Mice were subsequently infected with 10-20 plaque forming units of mouse adapted Influenza A virus and harvested 9 days post infection. Data includes bronchoalveolar lavage (BAL) cell count, daily weight change, and flow cytometry analysis of lung cells. No statistical significance in BAL cell count of mice air or cannabis smoke exposed was seen. Air exposed female mice exhibited significantly more weight loss than cannabis exposed female mice, indicating a more severe response to the virus. Cannabis exposed mice exhibited less weight loss on days 5 to 9. Continuing studies include a more detailed flow analysis of cannabis smoke exposed immune cells and its impact on viral infection.

Standard diagnostic tests for common bacterial and viral infections are invasive and uncomfortable, especially for children. Difficulty performing these tests leads to diseases such as strep throat or pneumonia going undiagnosed in children with significant health risks. There is a need for more accessible diagnostics. Our research uses saliva analysis as a diagnostic medium for Streptococcus pyogenes. This presentation depicts a novel saliva collection platform that is designed and engineered to be child-friendly, effective at pathogen collection, and is suitable for home use. We produced prototype devices using 3D printing, computer numerical control (CNC milling), and molding. Human studies were performed to examine usability and inform further design. In addition to engineering the device, we tested coatings and capture of bacteria. Preliminary data finds success in capturing S. pyogenes. Collection of commensal oral bacteria capture is the next step of our human studies. Our expectation is to find success with our device in a human mouth. Once our device and system are established, we plan to expand to include additional diseases. We will be presenting our innovations in the device as well as our preliminary data on the efficient capture of bacteria. We hope this technology will provide diagnostics with a way to provide care to underserved populations as well as help us to better collect data regarding the bacteria and viruses present in previously inaccessible populations. 

Toll-interacting protein (TOLLIP) is a ubiquitin-binding protein that is involved in the signaling pathways of Interleukin-1 receptor (IL-1R) and Toll-like receptors (TLR). Previous studies have shown that single nucleotide polymorphisms in the TOLLIP gene are associated with mortality and outcomes in idiopathic pulmonary fibrosis, however, the biology of TOLLIP in lung injury and repair is unknown. While animal studies have shown that TOLLIP modulates acute inflammation, the effect of TOLLIP deficiency in the mouse model of lung injury and fibrosis is unknown. In this study, our group investigated the effect of TOLLIP deficiency in the bleomycin model of lung injury. Previously, we observed that TOLLIP deficiency attenuated acute lung injury in the lipopolysaccharide (LPS) model of lung injury. We hypothesize that TOLLIP deficiency leads to increased lung injury in bleomycin-induced lung injury. To study this, we treated wild-type (WT) and TOLLIP knockout (KO) mice with bleomycin through intratracheal instillation (IT). At 14 days post-treatment, we collected bronchial alveolar lavage fluid (BALF) and lung tissues to evaluate the degrees of lung injury and fibrosis. Throughout this study, I helped maintain the mouse colonies, genotype the mice, process the samples during the harvests, collect BALF cell differentials, and analyze the data. Preliminary analyses of weight loss, BALF total protein, and cell differentials suggest TOLLIP deficiency results in worse lung injury at 14 days post-bleomycin. This work provides insight into the role of TOLLIP deficiency as an attenuator in long-term lung injuries and how it may be used as a potential treatment for inflammatory disorders and infections.

Dental implantation is a common clinical procedure used to replace missing teeth and maintain bone structure and facial aesthetics. However, it leads to unexpected side effects, including bone loss or peri-implantitis in 1 out of 10 cases due to failure of osseointegration, defined as improper integration of the implant into the mineralized bone. To enhance osseointegration, the present study aimed to form a layer of hydroxyapatite that could facilitate the integration of the implant (Titanium or Zirconia) with the alveolar bone that lead, while also having antimicrobial property to prevent local infection. Our previous study demonstrated that titanium-binding peptides (TiBPs) are able to bind specifically to the surface of Ti and that amelogenin-derived peptide (ADPs) can be used for direct remineralization on the bone surface. We also identified antimicrobial peptides (AMPs) that can inhibit common oral bacterial growth. These results imply that there is an opportunity to design two of heterofunctional peptides, both binding to Ti with one has the function of directing biomimetic remineralization process, while the other providing antimicrobial activity. The Ti-surface is modified by chimerizing the TiBPs and the ADPs, and TiBPs and AMPs, with short amino acid sequences. The overall process is separated into two main steps: 1. Designing and synthesizing the chimeric peptide; 2. Characterizing (a) binding, (b) mineralization and (c) antimicrobial efficacy of chimeric peptides on the implant surface. We predict that the chimeric peptides will have high binding affinity to the titanium surface while simultaneously enabling mineralization on the implant surface and inhibiting the growth of bacteria. The present aims to contribute to the foundation of finding a long-term novel dental implant treatment via the molecular biomimetic approach towards a clinical strategy to enhance the long-term durability of dental implants. The research is supported by Mary Gates Scholarship (YL), Spencer Funds from School of Dentistry, and CoMotion.

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.

Cell quiescence is defined as the reversible state of a cell in which it does not divide but retains the ability to re-enter cell proliferation. Quiescence can either be programmed or injury-induced. The phenomenon is influenced by mTORC1 signaling, a target for cancer therapeutics. Improved understanding of the mechanisms behind stem cell quiescence holds potential for future therapeutics against tumorigenesis. Cancer biology research in the past have suggested a correlation between mTORC1 activity and mitochondrial fusion and biogenesis. To pinpoint how mitochondrial dynamics influence stem cell quiescence, I radiated, dissected, and imaged UAS-Gal4 Drosophila models with specific mitochondrial gene knockdowns. As hypothesized, Drosophila ovaries with mitochondrial biogenesis and inner-membrane fusion knockdowns failed to exit quiescence after radiation. To further confirm our hypothesis, I am now directly quantifying the mTORC1 activity of mitochondrial knockdown Drosophila lines. Once collected, our data will contribute to ongoing research in cancer therapeutics.

Antibiotic treatment often fails in chronic bacterial infections; many resistant bacteria no longer respond to antibiotic treatments once designed to eliminate them. The chronic infections of bacteria such as Pseudomonas aeruginosa (Pa) that afflict people with cystic fibrosis (CF) are a prime example. In CF, standard antibiotic susceptibility testing does not accurately predict treatment efficacy, and genetically unstable resistance is one potential explanation. Genetically unstable resistance occurs when resistance-producing mutations are rapidly lost or compensated for in the absence of antibiotics. Clinical susceptibility testing likely fails to detect unstable resistance as many bacterial growth generations (in the absence of antibiotics) are used in preparing isolates for testing. We hypothesized that Pa cultured from the lungs of tobramycin-treated CF patients exhibit unstable antibiotic resistance that rapidly reverts upon growth in the absence of antibiotic pressure. To test this, we cultured Pa isolates from patients who are being treated with antibiotics in a manner that maintains antibiotic selection during growth steps. We then measured their resistance levels before and after growth in the absence of antibiotics to identify isolates with unstable resistance. Whole genome sequencing of sensitive and resistant isolate pairs identified the genetic mechanisms causing unstable resistance. Our data shows that individual isolates from many patients exhibit a wide range of instability. Some isolates exhibited as much as an over a hundred-fold decrease in inhibitory concentration after antibiotic-free growth, while other isolates from the same sample had stable resistance. This work could lead to new sequenced-based methods to detect unstable resistance in patients, new approaches to select antibiotics for treatment and a greater understanding of antibiotic efficacy in chronic infections.