The gut microbiome plays a significant role in nerve signaling through sensory neurons. Peripheral sensory neurons are hypothesized to receive signals from the gut microbiome and deliver them to the brain, depicting how the microbiome influences host physiology. Notably, it has been found that the brain’s nucleus of the solitary tract (NTS) receives inputs from peripheral sensory neurons. The purpose of this project was to determine whether gut microbiome-depleting antibiotics affect NTS activation by depleting the microbiome or signaling to the NTS itself. Individual antibiotics were utilized to selectively deplete specific gut microbial strains. This was done by administering the antibiotics (metronidazole or ampicillin) or vehicle solution. Fecal samples from each mouse in each group were then collected, after which they were homogenized and gavaged into germ-free mice. Cardiac perfusions were then conducted, and the brains were collected and frozen for cryosectioning. Immunohistochemistry staining was then conducted to stain for activated neurons. The tissue samples were then imaged through confocal microscopy and computationally analyzed to individually count neurons. The results depict how there is no significant difference in cFos+ neurons between mice administered antibiotic FMT and mice administered vehicle FMT, indicating that the antibiotics themselves may be signaling to the NTS. Through this, the impact of gut microbiome depletion by antibiotics on behavioral responses can be determined.

Alzheimer’s disease (AD) is a neurodegenerative disorder characterized by extracellular amyloid-β (Aβ) plaque accumulation, intracellular tau pathology, and chronic neuroinflammation. Aβ peptides are generated through aberrant cleavage of amyloid precursor protein, leading to aggregation and disruption of neuronal communication. Although FDA-approved antibodies such as Lecanemab and Donanemab reduce amyloid burden, their efficacy is limited by poor blood–brain barrier penetration, repeated dosing, and potential exacerbation of neuroinflammation. To address these limitations, chimeric antigen receptor macrophages (CAR-Ms) combine antigen-specific recognition with enhanced phagocytic signaling. To enable a self-renewing therapy, CAR constructs can be introduced into hematopoietic stem cells (HSCs), allowing sustained in vivo generation of CAR-expressing macrophages. We engineered anti-Aβ CAR constructs derived from Lecanemab and Donanemab and evaluated intracellular signaling domains. CARs incorporating single-chain variable fragments (scFvs) were designed with optimized hinge and transmembrane regions, including CD8 or Fc-associated domains, alongside CD3ζ, FcRγ, and FcγRIIa signaling domains. Flow cytometry revealed that CARs with CD8 transmembrane domains exhibited superior Aβ binding compared to Fc-derived variants, a finding validated in lentivirally transduced THP-1 cells. In vivo bone marrow studies demonstrated robust engraftment and differentiation of CAR-expressing macrophages from HSCs.

Solar erythema, more commonly known as sunburns, is an acute inflammatory skin reaction that affects 1 in 3 US adults each year, posing a significant risk factor for skin cancer. Previous research found that sunburns appeared earlier and resulted in more severe symptoms in female than male mice. Our study aims to investigate whether this disparity in symptoms persists when specific immune system genes and receptors are knocked out. Data was collected using wild type (WT), YAP/TAZ lysozyme M (LysM), and X-C chemokine receptor 1 (XCR1) mice. The YAP/TAZ LysM mice contained myeloid cells deficient in transcriptional coactivators, while the XCR1 mice were injected with diphtheria toxin and have depleted XCR1 dendritic cells. Sunburns were induced by exposing the mice to UVB rays for ten minutes per day over a course of three days. Images were taken of the mice each day to document changes in skin redness and swelling. The images showed increased redness and swelling starting on Day 2 in the female WT and LysM YAP/TAZ mice groups. Consistent with the images, the WT and YAP/TAZ LysM female mice had higher average myeloperoxidase (MPO) activity, indicating greater levels of inflammation. Interestingly, average MPO activity was nearly equivalent for the male and female XCR1 mice. Female WT mice also had the greatest level of inflammation compared to other mice groups. As we continue to study sex differences in sunburns, we can pave the way for innovative therapies to treat solar erythema that can target specific immune mechanisms.

Current hematopoietic stem cell (HSC) gene therapies using electroporation to genetically engineer cells ex vivo result in cells exiting quiescence, reducing their ability to self-renew and their long-term engraftment potential in a patient once infused. Ex vivo gene therapy strategies also often require cytotoxic bone marrow conditioning. An alternative to these ex vivo approaches is to package gene editing reagents into lipid nanoparticles (LNPs) to edit HSCs directly in vivo. To best reach HSCs with the highest self-renewal potential, we are designing and testing LNPs configured to target the CD90 surface marker. Using CD90-Positive (CD90+) Jurkat cells as an HSC model cell line, we tested CD90-antibody-conjugated LNPs (CD90-LNPs) loaded with mRNA encoding for green fluorescent protein (GFP). We observed higher expression of GFP upon treatment with CD90-LNPs compared to LNPs lacking CD90 antibodies. Additionally, LNPs formulated at an amine to phosphate (NP) ratio of 16 with the ionizable lipid C14-4 induced the highest editing efficiency. However, one downside to LNP treatment in vivo is the potential for off target transfection of off-target cell types. Recent work has demonstrated that LNPs formulated with DSPE-PEG 2000 and monoclonal antibodies only result in transfection of cells expressing the target of the antibodies. Leveraging this insight, our future studies will investigate whether NP 16 LNPs formulated with C14-4, DSPE-PEG 2000, and CD90 antibodies can achieve effective editing exclusively in CD90+ HSCs.

A scientific manuscript often selectively reveals a story of clear-cut success, hiding the roadblocks that are regularly faced by researchers. This presentation seeks to provide an authentic view of science, recounting the hurdles faced during my research project as well as the ways my lab and I have sought to overcome them. To treat HPV-associated cancer, which comprises 5% of all human malignancies, my lab seeks to modify hematopoietic stem cells with a T cell receptor recognizing HPV16’s E7 oncoprotein. Engraftment of HSCs in patients can generate sustained production of antigen-specific T cells to combat HPV16+ tumors. Initial attempts at developing this therapy were functional in vitro but failed to potentiate E7 TCR reconstitution in mice, which we hypothesize to be the result of an excessively strong MND promoter interfering with T cell development. Hence, we’ve developed two new constructs driven by EF1a and PGK promoter to test these as alternatives to MND. While PGK lentiviral vectors were capable of transducing primary T cells, EF1a lentivirus initially facilitated low TCR expression, requiring further analysis and optimization of vector production. Moreover, a coincubation assay measuring E7 TCR+ cytokine expression in response to target cells was refined to better demonstrate effector function after deeper consideration of cytokine expression kinetics. This project showcases both the potential for an HSC-based TCR therapy against HPV16+ malignancy as well as the many Plan Bs developed during a research project.