HIV remains a major public health issue in spite of antiretroviral therapy (ART). An innovative vaccine that can induce long-lasting and effective immunity is required to curb the persistently high numbers of new infections worldwide.
A novel DNA vaccine was generated using a Simian-Human Immunodeficiency Virus (SHIV) backbone with a Zambian T/F clade C envelope and under the control of the caprine arthritis encephalitis virus long terminal repeats (LTRs) for constitutive expression. Due to the deleted integrase, this DNA vaccine "CSH-DIN-T/F Z331" performs only a single replication cycle. To increase immunogenicity, the co-expression of apoptotic genes (BAX, BAK, or caspase 8) incorporated at the end of Pol was tested to promote the release of apoptotic bodies taken up by dendritic cells leading to cross-presentation of antigen. The three vaccines (CSH-DIN-T/F Z331-BAX, CSH-DIN-T/F Z331-BAK, and CSH-DIN-T/F Z331-Cas8) were tested in vitro for expression and in vivo in BALB/cJ mice for immunogenicity.
Transduced HEK293 cells co-cultured with CEMx174 confirmed the single replication cycle of the DNA vaccine and the induction of apoptosis by CSH-DIN-T/F Z331-Cas8 based on Annexin V expression. BALB/cJ mice were immunized with a combined intramuscular + intradermal/electroporation approach. Intracellular cytokine staining (ICS) from splenocytes collected 12 weeks post-prime/6 weeks post-boost demonstrated a clear superiority of caspase 8 expressing construct over the others, with higher proportions of IFN-γ-, IL-2-, and IL-21-producing CD8 T cells specific to Env, Gag, and Nef. The kinetics of immune response after various immunization schedules were also investigated.
This novel single-cycle DNA vaccine with apoptotic genes demonstrated an enhanced immunogenicity primarily for antigen-specific CD8+ T-cell responses.
Copyright © 2025 Bose, Rogers, Shirreff, Chebloune and Villinger.

The regulation of Cd4 expression during T-cell development and immune responses is essential for proper lineage commitment and function in the periphery. However, the mechanisms of genetic and epigenetic regulation are complex, and their interplay not entirely understood. Previously, we demonstrated the need for CD4 upregulation during positive selection to ensure faithful commitment of MHC-II-restricted T cells to the CD4 lineage. In this study, we investigate whether a conserved region, here called NCE, that is proximal to the Cd4 silencer and contains E4m has the required developmental-stage-specific canonical enhancer function and TCR responsiveness to mediate the CD4 upregulation required to prevent lineage errors.
To investigate the role of NCE, transient transfection of reporter plasmids was performed in thymoma cell lines arrested at the double-positive (DP, CD4+CD8+) and intermediate (INT, CD4+CD8lo) stages of development. CRISPR/Cas9-mediated deletion of the coreNCE/E4m region was carried out in these cell lines to assess its impact on CD4 surface expression, re-expression rates, and TCR signaling responsiveness. To avoid developmental alterations from direct manipulation of the endogenous Cd4 locus in vivo, BAC-transgenic reporter mice were generated with the locus modified to express EGFP in the presence or absence of NCE. EGFP mRNA levels were measured via RT-qPCR, and EGFP fluorescence was analyzed in post-selection thymocytes.
Our in vitro experiments demonstrate that NCE by itself can function as an enhancer at the INT, but not the DP stage of development. Furthermore, CRISPR/Cas9-mediated deletion of coreNCE/E4m resulted in reduced CD4 surface levels, slower re-expression rates, and reduced TCR signaling responsiveness in INT cells, but not in DP cells. In vivo, NCE-sufficient transgenic mice exhibited upregulation of Cd4 reporter EGFP mRNA levels at the INT stage and a corresponding upregulation of EGFP fluorescence, whereas NCE-deficient mice showed a significant loss of Cd4 reporter EGFP mRNA and no detectable EGFP production in any post-selection thymocytes.
This study demonstrates that the canonical enhancer function of coreNCE/E4m is essential for CD4 upregulation following positive selection. The NCE region, with its developmental-stage-specific activity and its known epigenetic regulatory capabilities, ensures faithful lineage commitment to the CD4 lineage.
Copyright © 2025 Swan, Fujii, Guzynski, Page, Meyers, Penev, Littleton, Azzahra, Richardson and Sarafova.

Low-grade inflammation and pathological endochondral ossification are key processes underlying the progression of osteoarthritis, the most prevalent joint disease worldwide. In this study, we employed a multi-faceted approach, integrating publicly available datasets, in silico analyses, in vitro experiments and in vivo models to identify new therapeutic candidates targeting these processes. Data mining of transcriptomic datasets identified EPHA2, a receptor tyrosine kinase associated with cancer, as being linked to both inflammation and endochondral ossification in osteoarthritis. A computational model of cellular signaling networks in chondrocytes predicted that in silico activation of EPHA2 in healthy chondrocytes increases inflammatory mediators and induces hypertrophic differentiation, a hallmark of endochondral ossification. We then evaluated the effect of EPHA2 inhibition using the tyrosine kinase inhibitor ALW-II-41-27 in cultured human chondrocytes from individuals with osteoarthritis, demonstrating significant reductions in both inflammation and hypertrophy. Additionally, systemic subcutaneous administration of ALW-II-41-27 in a mouse osteoarthritic model attenuated joint degeneration by reducing local inflammation and pathological endochondral ossification. Collectively, this study demonstrates a novel drug discovery pipeline that integrates computational, experimental, and animal models, paving the way for the development of disease-modifying treatments for osteoarthritis.
© 2024 The Authors.

Nanoparticles are increasingly being used in medicine, cosmetics, food, and manufacturing. However, potential toxicity may limit the use of newly engineered nanoparticles. Prior studies have identified particle characteristics that are predictive of toxicity, although the mechanisms responsible for toxicity remain largely unknown. Nanoparticle treatment in cell culture, combined with high-throughput chemical screen allows for systematic perturbations of thousands of molecular targets against potential pathways of toxicity. The resulting data matrix, called chemical compendium, can provide insights into the mechanism of toxicity as well as help classify nanoparticles based on toxicity pathway.
We performed unbiased screens of 1280 bioactive chemicals against a panel of four particles, searching for inhibitors of macrophage toxicity. Our hit compounds clustered upon inhibitors of kinases involved in phagocytosis, including focal adhesion kinase (FAK), with varying specificity depending on particles. Interestingly, known inhibitors of cell death including NLRP3 inflammasome inhibitor were unable to suppress particle-induced macrophage death for many of the particles. By searching for upstream receptors of kinases, we identified Cd11b as one of the receptors involved in recognizing a subset of particles. We subsequently used these hit compounds and antibodies to further characterize a larger panel of particles and identified hydrodynamic size as an important particle characteristic in Cd11b-mediated particle uptake and toxicity.
Our chemical compendium and workflow can be expanded across cell types and assays to characterize the toxicity mechanism of newly engineered nanoparticles. The data in their current form can also be analyzed to help design future high-throughput screening for nanoparticle toxicity.
© 2024. The Author(s).

Neutrophils play an essential role in Alzheimer's disease (AD) pathology. However, the extent of their heterogeneity remains poorly explored, particularly in the context of developing novel therapies targeting these cells.
We investigate the population structure of neutrophils purified from peripheral blood samples of AD mice. Utilizing single cell RNA sequencing, we comprehensively map neutrophil populations into six distinct clusters and find that the Neu-5 subset is specially enriched in AD mice. This subset exhibits fewer specific granules and a lower mature score. Gene ontology (GO) analysis reveals that genes involved in cytokine-mediated signaling are downregulated in the Neu-5 cluster. Furthermore, we identify the Ccrl2 gene is specifically upregulated in this subgroup, which is confirmed by flow cytometry in AD mice. Finally, immunohistochemical staining indicates that CCRL2 protein is increased in the brains of AD mice.
We identify a unique CCRL2 positive neutrophil cluster, that is specifically enriched in the peripheral blood of AD mice.
© 2024. The Author(s).