Spatially visualized single-cell pathology of highly multiplexed protein profiles in health and disease

Deep molecular profiling of biological tissues is an indicator of health and disease. We used imaging mass cytometry (IMC) to acquire spatially resolved 20-plex protein data in tissue sections from normal and chronic tonsillitis cases. We present SpatialViz, a suite of algorithms to explore spatial relationships in multiplexed tissue images by visualizing and quantifying single-cell granularity and anatomical complexity in diverse multiplexed tissue imaging data. Single-cell and spatial maps confirmed that CD68+ cells were correlated with the enhanced Granzyme B expression and CD3+ cells exhibited enrichment of CD4+ phenotype in chronic tonsillitis. SpatialViz revealed morphological distributions of cellular organizations in distinct anatomical areas, spatially resolved single-cell associations across anatomical categories, and distance maps between the markers. Spatial topographic maps showed the unique organization of different tissue layers. The spatial reference framework generated network-based comparisons of multiplex data from healthy and diseased tonsils. SpatialViz is broadly applicable to multiplexed tissue biology.

Evaluation of Viability and Proliferation Profiles on Macrophages Treated with Silica Nanoparticles In Vitro via Plate-Based, Flow Cytometry, and Coulter Counter Assays

Nanoparticles (NPs) are known to interfere with many high-throughput cell viability and cell proliferation assays, which complicates the assessment of their potential toxic effects. The aim of this study was to compare viability and proliferation results for colloidal silica (SiO2 NP; 7 nm) in the RAW 264.7 mouse macrophage cell line using three different techniques: plate-based assays, flow cytometry analysis, and Coulter counter assays. Our data indicate that CellTiter-Blue, XTT, and CyQuant plate-based assays show increased values over control at low SiO2 NPs concentrations (0.001–0.01 g/L). SiO2 NPs show little-to-no interference with flow cytometry and Coulter counter assays, which not only were more reliable in determining cell viability and proliferation at low concentrations in vitro, but also identified changes in cell granularity and size that were not captured by the plate-based assays. At high SiO2 NP concentrations (1 g/L) all techniques indicated cytotoxicity. In conclusion, flow cytometry and Coulter counter identified new cellular features, and flow cytometry offered more flexibility in analyzing the viability and proliferation profile of SiO2 NP-treated RAW 264.7 cells.

Heterologous processing of rat prosomatostatin to somatostatin-14 by PC2: requirement for secretory cell but not the secretion granule

The role of PC2 in prosomatostatin (PSS) processing was investigated in GH3/GH4C1 pituitary cells. These cells are sparsely granulated, express different amounts of PC2 and no PC1. We described heterologous processing of rat PSS (rPSS) co-expressed with PC2 in stably transfected cells, correlate PC2 protein levels under different conditions of transfection with efficiency of PSS processing to somatostatin-14 (SS-14), determine the effect of modulating cell granularity on enzyme expression and PSS processing, and compare the relative potency of PC2 with that of PC1, PSS and cleavage products were monitored by HPLC and radioimmunoassay of SS-like immunoreactivity (SSLI). Radioimmunoassay analysis of N-terminal PC2-like immunoreactivity (PC2 LI) in GH4C1:rPSS, GH4C1:rPSS + PC2 and GH3:rPSS transfectants showed a gradient of PC2 protein of 1:2.6:3.4 in cell extracts and 1:4.7:9 in secretion media from these cells respectively. The concentration of PC2 protein correlated with SS-14 conversion efficiency was 36 +/- 3% in GH4C1:rPSS cells, 56 +/- 7% in GH4C1:rPSS-PC2 cells and 100% in GH3:rPSS cells. Treatment of GH4C1:rPSS + PC2 cells with epidermal growth factor, insulin, and beta-estradiol to induce granules, significantly increased basal and forskolin-stimulated co-release of SS LI and PC2 LI, but had no influence on SS-14 processing efficiency. Hormone treatment led to a small increase in the ratio of mature PC2 (68 kDa) to proPC2 (75 kDa) forms. PC1 stably transfected in GH4C1 cells produced significantly greater SS-14 conversion (62% in cells, 66% in media) compared with PC2 transfectants (53% in cells, 47% in media) These results provide the first proof that PC2 can effect dibasic processing of mammalian PSS, and, along with PC1, qualifies as an authentic SS-14 convertase. The activity of PC2 requires the milieu of the secretory cell but not the secretory granule.

Effects of high temperature and acute diuresis on glucose-6-phosphate dehydrogenase activity of macula densa and plasma renin activity in rats

Experiments performed in rats exposed to heat and to acute diuresis revealed: (a) a positive influence of the environmental temperature on plasma renin activity; (b) a dissociation between plasma renin activity and the glucose-6-phosphate dehydrogenase activity of the macula densa following furosemide administration; and (c) the absence of any significant changes in juxtaglomerular cell granularity despite a very significant increase in plasma renin activity.

Experimental Studies on Erythropoietin. II. The Relationship between Juxtaglomerular Cells and Erythropoietin

The relationship between erythropoietin production and the juxtaglomerular (JG) cells of the kidney were studied using Harada’s method for staining and Hartroft’s method for estimating JG granularity. In stimulated erythropoiesis (after bleeding and phenylhydrazine injection), the JG cells also showed hypergranularity. In depressed erythropoiesis, on the contrary, the JG cells showed hypogranularity. Furthermore, from the observation of the chronological change of the JG cell, granularity tended to change in parallel with erythropoietin production. This study gave an indirect evidence of the secretion of erythropoietin by juxtaglomerular cells of the kidney.