Cytotoxic T lymphocytes (CTLs) are crucial in killing infected cells by recognizing antigens presented on Major Histocompatibility Complex class I molecules (MHC-1) and triggering apoptosis. However, B-cell malignancies and Cytomegalovirus infection evade CTLs by disrupting MHC-1. Chimeric Antigen Receptors (CAR) address this problem by overcoming the MHC-1 dependence. However, traditional CAR relies on a non-physiological CD3 zeta chain linkage in its signaling domain, leading to tonic signaling and T-cell exhaustion. On the other hand, the antibody-𝛄/𝛅 CAR we developed incorporates an antigen-binding domain derived from antibody variable light and variable heavy domains (targeting CD19 for B-cell malignancies and 21E9 for CMV infection) and a constant region derived from a native 𝛄/𝛅 T-cell receptor that doesn’t require a CD3 complex, unlike conventional CARs. Here, we constructed two CARs based on CD19 and 21E9, and provided preliminary data on molecular cloning steps: PCR amplification, restriction digestion, infusion cloning, bacterial transformation, culture expansion, DNA isolation, and expression. In the next steps, we will produce lentivirus by transfecting LentiX cells to transduce CD8+ T-cells to perform functional assays to assess cytotoxicity, cell proliferation, and tonic signaling. If successful, the new antibody-𝛄/𝛅 CAR could improve CAR-T durability and therapeutic efficacy.

Antimicrobial resistance (AMR) is a growing threat to food safety and public health. Retail meat, especially commodities derived from treated animals, may serve as reservoirs for resistant bacteria, including Enterococcus spp. This study evaluated prevalence and AMR trends in Enterococcus spp. from retail pork collected in California through the NARMS Retail Food Surveillance program. Retail pork samples were collected in 2018, 2020, 2022, and 2024 (n=214) and processed using standard NARMS protocols. Antimicrobial susceptibility was determined by broth microdilution and minimum inhibitory concentration (MIC) results were interpreted using published NARMS criteria. Overall, 169/214 (78.9%) samples were Enterococcus-positive, with isolation rates of 83.9% (2018), 72.0% (2020), 79.2% (2022), and 83.3% (2024), respectively. Among 93 isolates with MIC data available, all isolates showed resistance to ≥1 drug (100%), 97.9% to ≥2 drugs, and 71% were multidrug resistant (≥3 classes). Resistance was highest to erythromycin (81%) in all Enterococcus species and to quinupristin/dalfopristin (87.0%) in E. faecium. Enterococcus was moderately resistant to daptomycin (47%) and the resistance rates were similar in products with claims of reduced antibiotics (83%) and in conventional products (80%). No resistance was observed to linezolid or vancomycin. These findings demonstrate substantial AMR in Enterococcus spp. from retail pork and underscore the importance of continued surveillance.

Hepatocellular carcinoma (HCC) is the most common primary liver cancer and a leading cause of cancer-related mortality worldwide. The rising prevalence of metabolic dysfunction-associated steatohepatitis (MASH) has increased HCC incidence, yet the immune mechanisms underlying disease progression remain poorly understood. While IL27 receptor (IL27R) signaling has been found to suppress innate anti-tumor responses, its role in regulating adaptive immunity, particularly regulatory T cells (Tregs) in MASH-associated HCC development, remains unclear. To test this, we used a MUP-uPA mouse model of MASH-driven HCC with Treg-specific Il27ra deletion (Il27raflox Foxp3Cre MUP). Tumor burden was assessed by macroscopic analysis and histology, while proliferation, fibrosis, and immune composition were evaluated using IHC and qPCR. Loss of IL27R signaling in Tregs resulted in reduced tumor number and load, along with decreased tumor cell proliferation, shown by Ki67 staining and supported by reduced Ccnd1 expression. Mice lacking IL27R in Tregs also exhibited reduced collagen deposition in non-tumor liver tissue. Analysis of immune cells on histological sections showed lower accumulation of F4/80 macrophages in tumors of Il27raflox Foxp3Cre MUP mice as compared to controls, suggesting a link between Il27R-Treg activation and macrophage accumulation. These findings suggest that IL27R signaling in Tregs regulates pro-tumorigenic microenvironment controlling fibrosis, tumor cell proliferation, and macrophage accumulation.

Regulatory T cells (Tregs) are critical for maintaining immune homeostasis and preventing autoimmunity. Age-associated thymic involution contributes to declining immune regulation and increased susceptibility to autoimmunity and cancer. A new Treg population, thymic-recirculating Tregs (trTregs), arises from peripheral or thymic origins and re-enters the thymus, where it may increase in frequency with age. However, reliable markers for identifying human trTregs remain poorly defined. Our previous studies in mouse models identified candidate markers distinguishing thymic-naive Tregs (tnTregs) from trTregs through flow cytometry and T-cell receptor excision circle (TREC) analysis. Here, we extend these findings to human thymic tissue and peripheral blood mononuclear cells (PBMCs) to evaluate their translational relevance. Flow cytometry, fluorescence-activated cell sorting (FACS), and quantitative PCR (qPCR) were used to characterize marker expression and TREC levels across Treg subsets. Our findings support CD39 as a candidate surface marker of human trTregs, based on its increased representation in peripheral Tregs and sustained expression in thymic Tregs observed in recent screening experiments. In addition, thymic Treg phenotypes appeared relatively stable across samples from one month to three years of age. ddPCR and TREC analyses further identified markers that may distinguish the thymic Treg populations. Together, these findings contribute to define human trTregs and improve understanding of immune regulatory mechanisms

Alphaviruses are positive-sense, single-stranded RNA viruses transmitted to humans by mosquitoes. Their genomes encode structural and nonstructural polyproteins that must be cleaved into individual proteins for effective viral replication and transmission. These viral proteins are subject to host-mediated post-translational modifications, including phosphorylation. We propose that phosphorylation of viral proteins functions as a biosensor, modulating critical steps in the virus life cycle. To explore this, we analyzed phosphosites on the nonstructural proteins (nsPs) as potential regulators of viral replication complex formation. One site of particular interest is T5 on nsP2, located near the protein’s N-terminal cleavage site. We hypothesize that the phosphorylation state of T5 on nsP2 alters polyprotein cleavage and impacts viral RNA replication. To test this, we cloned the Sinbis virus (SINV) infectious clone plasmid pToto1101 with a FLAG-tag into nsP3 for fluorescent visualization of nonstructural polyprotein processing products. Additionally, we cloned a plasmid containing the biotin ligase TurboID conjugated to nsP2 to identify biotinylated proximity proteins. Mutating these plasmids at nsP2 T5 and transfecting them into HEK293T cells will highlight the effect of phosphorylation at T5 on the viral life cycle and indicate which kinases may be responsible. By studying how phosphorylation regulates polyprotein cleavage, we aim to uncover host-virus signaling interactions and identify targets for antiviral intervention.

Junin virus (JUNV), the etiological agent of Argentine hemorrhagic fever, remains a significant public health concern because, though the Candid#1 vaccine is available as a preventative measure, there remains no effective antiviral treatments after infection occurs. To advance the search for new therapies, in vitro infection models were established in both VeroE6 cells and primary HUVECs. The immunofluorescence assay for JUNV detection was optimized through systematic screening of monoclonal antibodies. This screening identified a highly effective ten-antibody cocktail that produced strong staining with low background across multiple infection levels. The optimized assay was used to run a drug screening of metabolite and kinase inhibiting compounds. The screen revealed numerous candidates with strong potential antiviral activity. Future studies will refine hit selection by validating candidate antivirals in additional cell types and animal models, developing complementary RT-qPCR methods for quantification, and investigating the mechanisms responsible for viral inhibition. Together, these results establish a robust platform for JUNV antiviral discovery and identify host kinase pathways as promising therapeutic targets.