Cuprolinic Blue: a specific dye for single-stranded RNA in the presence of magnesium chloride. II. Practical applications for light microscopy

The application of the new nucleic acid dye Cuprolinic Blue to cell smears and tissue sections has been described. Without added cations, Cuprolinic Blue stains both DNA and RNA, whereas in the presence of 1 M MgCl2, Cuprolinic Blue specifically stains single-stranded RNA only. The total RNA can be stained after removal of DNA by DNAase digestion. Fixation in a modified Carnoy solution gave optimal staining results in all cases tested. By cytophotometry, a reliable and reproducible relative estimate can be obtained of the total nucleic acid content, the total RNA content and the amount of single-stranded RNA alone per cell.     

Meningococcal meningitis: an atypical case

A case of meningococcal meningitis is described in a patient who had not received antibiotics before admission. Three lumbar punctures were performed over a seven-day period, but only the cerebrospinal fluid from the third yielded a positive culture. The importance of repeated lumbar punctures in patients with undiagnosed meningitis is emphasized.     

Pronase digestion of amino-acid binding components on the surface of Bacillus subtilis cells and minicells

The addition of 250 μg/ml pronase to either cells or minicells of Bacillus subtilis does not interfere with growth, macromolecular synthesis or maintenance of cell integrity. Radioactive amino acids such as proline or isoleucine taken up by cells or minicells are bound to surface components which prevent their release by washing. Pronase treatment causes the release of a significant percentage of these bound amino acids. In cells and minicells exposed to pronase before the addition of labeled amino acids, initial binding capacities are greatly reduced. These data suggest a new approach to the investigation of amino acid binding components on the surface of Bacillus subtilis.     

B7-H3 drives immunosuppression and Co-targeting with CD47 is a new therapeutic strategy in β-catenin activated melanomas

In melanoma, immune cell infiltration into the tumor is associated with better patient outcomes and response to immunotherapy. T-cell non-inflamed tumors (cold tumors) are associated with tumor cell-intrinsic Wnt/β-catenin activation, and are typically resistant to anti-PD-1 alone or in combination with anti-CTLA-4 therapy. Reversal of the ‘cold tumor’ phenotype and identifying new effective immunotherapies are challenges. We sought to investigate the role of a newer immunotherapy agent, B7-H3, in this setting. RNA sequencing was used to identify co-targeting strategies upon B7-H3 inhibition in a well-defined preclinical melanoma model driven by β-catenin. We found that immune checkpoint molecule B7-H3 confers a suppressive tumor microenvironment by modulating antiviral signals and innate immunity. B7-H3 inhibition led to an inflamed microenvironment, up-regulation of CD47/SIRPa signaling, and together with blockade of the macrophage checkpoint CD47 resulted in additive antitumor responses. We found that the antitumor effects of the B7-H3/CD47 antibody combination were dependent on cytokine signaling pathways (CCR5/CCL5 and IL4).