A Hitchhiker's guide to high-dimensional tissue imaging with multiplexed ion beam imaging.

Advancements in multiplexed tissue imaging technologies are vital in shaping our understanding of tissue microenvironmental influences in disease contexts. These technologies now allow us to relate the phenotype of individual cells to their higher-order roles in tissue organization and function. Multiplexed Ion Beam Imaging (MIBI) is one of such technologies, which uses metal isotope-labeled antibodies and secondary ion mass spectrometry (SIMS) to image more than 40 protein markers simultaneously within a single tissue section. Here, we describe an optimized MIBI workflow for high-plex analysis of Formalin-Fixed Paraffin-Embedded (FFPE) tissues following antigen retrieval, metal isotope-conjugated antibody staining, imaging using the MIBI instrument, and subsequent data processing and analysis. While this workflow is focused on imaging human FFPE samples using the MIBI, this workflow can be easily extended to model systems, biological questions, and multiplexed imaging modalities.

Epstein-Barr Virus Orchestrates Spatial Reorganization and Immunomodulation within the Classic Hodgkin Lymphoma Tumor Microenvironment.

Classic Hodgkin Lymphoma (cHL) is a tumor composed of rare malignant Hodgkin and Reed-Sternberg (HRS) cells nested within a T-cell rich inflammatory immune infiltrate. cHL is associated with Epstein-Barr Virus (EBV) in 25% of cases. The specific contributions of EBV to the pathogenesis of cHL remain largely unknown, in part due to technical barriers in dissecting the tumor microenvironment (TME) in high detail. Herein, we applied multiplexed ion beam imaging (MIBI) spatial pro-teomics on 6 EBV-positive and 14 EBV-negative cHL samples. We identify key TME features that distinguish between EBV-positive and EBV-negative cHL, including the relative predominance of memory CD8 T cells and increased T-cell dysfunction as a function of spatial proximity to HRS cells. Building upon a larger multi-institutional cohort of 22 EBV-positive and 24 EBV-negative cHL samples, we orthogonally validated our findings through a spatial multi-omics approach, coupling whole transcriptome capture with antibody-defined cell types for tu-mor and T-cell populations within the cHL TME. We delineate contrasting transcriptomic immunological signatures between EBV-positive and EBV-negative cases that differently impact HRS cell proliferation, tumor-immune interactions, and mecha-nisms of T-cell dysregulation and dysfunction. Our multi-modal framework enabled a comprehensive dissection of EBV-linked reorganization and immune evasion within the cHL TME, and highlighted the need to elucidate the cellular and molecular fac-tors of virus-associated tumors, with potential for targeted therapeutic strategies.

Characterization of Two New Multidrug-Resistant Strains of Mycobacterium smegmatis: Tools for Routine In Vitro Screening of Novel Anti-Mycobacterial Agents.

Mycobacterium tuberculosis is a pathogen of global public health concern. This threat is exacerbated by the emergence of multidrug-resistant and extremely-drug-resistant strains of the pathogen. We have obtained two distinct clones of multidrug-resistant Mycobacterium smegmatis after gradual exposure of Mycobacterium smegmatis mc² 155 to increasing concentrations of erythromycin. The resulting resistant strains of Mycobacterium smegmatis exhibited robust viability in the presence of high concentrations of erythromycin and were found to be resistant to a wide range of other antimicrobials. They also displayed a unique growth phenotype in comparison to the parental drug-susceptible Mycobacterium smegmatis mc² 155, and a distinct colony morphology in the presence of cholesterol. We propose that these two multidrug-resistant clones of Mycobacterium smegmatis could be used as model organisms at the inceptive phase of routine in vitro screening of novel antimicrobial agents targeted against multidrug-resistant Mycobacterial tuberculosis.

Acute and Subchronic Toxicity Studies of Aqueous Extract of Desmodium adscendens (Sw) DC.

Extracts of Desmodium adscendens (Sw) DC are used for the treatment of various diseases but limited toxicological evaluations have been done on the medicinal plant. This study investigates toxicity effects of the leave extract of D adscendens, and the possibility of drug-drug interaction of the plant extract when co-administered with other drugs. Oral administrations of leaf extract of D adscendens to white Wistar rats in an acute toxicity studies allowed the estimation of an LD50 (median lethal dose) value of 1122 mg/kg body weight. In a subchronic toxicity studies, the plant extract caused a decrease in zoxazolamine paralysis time and prevented thiopentone from causing sleep in test animals compared to controls. Overall, the results are consistent with the plant extract being safe at the doses administered in humans. However, the induction of the CYP enzymes is an indication of a possible drug interaction when the plant extract is co-administered with other drugs.