Cathepsin D: an Mϕ-derived factor mediating increased endothelial cell permeability with implications for alteration of the blood-retinal barrier in diabetic retinopathy.

Inflammation plays an important role in the pathogenesis of diabetic retinopathy (DR). We have previously reported increased monocyte (Mono) trafficking into the retinas of diabetic animals. In this study, we have examined the effect of activated Monos on retinal endothelial cells (ECs). The U937 Mϕ-conditioned medium (CM) significantly decreased the transendothelial resistance of EC monolayers as measured by electric cell-substrate impedance sensing (P= 0.007). The CM was fractioned, and the effective fraction (30-100 kDa) was analyzed by liquid chromatography-mass spectrometry, and cathepsin D (CD) was identified as a major secreted product. Immunoprecipitated CD resulted in decreased resistance in ECs (P= 0.006). The specificity of CD in mediating alterations of the EC barrier was confirmed using small interfering RNA. The decreased resistance correlated with a significantly increased gap between ECs. CD altered the Ras homolog gene family, member A/Rho-associated kinase pathway with increased stress actin filament formation in the EC layer. Increased CD levels were found in the retinas of diabetic mice (3-fold) and serum samples of patients with diabetic macular edema (1.6-fold) measured by Western blot and ELISA. These findings suggest an important role for Mϕ-derived CD in altering the blood-retinal barrier and reveal a potential therapeutic target in the treatment of DR.-Monickaraj, F., McGuire, P. G., Nitta, C. F., Ghosh, K., Das, A. Cathepsin D: an Mϕ-derived factor mediating increased endothelial cell permeability with implications for alteration of the blood-retinal barrier in diabetic retinopathy.

A rapid and high-throughput T cell immunophenotyping assay for cellular therapy bioprocess using the Cellaca® PLX image cytometer.

In cellular therapies chimeric antigen receptor (CAR) T or NK cells undergo phenotypic analysis at multiple stages during discovery and development of novel therapies. Patient samples are routinely analyzed via flow cytometry for population identification and distribution of CD3, CD4, and CD8 positive T cells. As an alternative or orthogonal method, image cytometry systems have been used to perform simple cell-based assays in lieu of flow cytometry. Recently, a new image cytometry system, the Cellaca® PLX (Revvity Health Sciences, Inc., Lawrence, MA), was developed for high-throughput cell counting and viability, immunophenotyping, transfection/transduction efficiency, and cell health assays. This novel instrument allows investigators to quickly assess several critical quality attributes (CQAs) such as cell identity, viability, and other relevant biological functions recommended by the International Organization for Standardization using the ISO Cell Characterization documents focused on cellular therapeutic products. In this work, we demonstrate a rapid and high-throughput image cytometry detection method for cellular immunophenotyping and viability using the Cellaca PLX system for samples throughout the cellular therapy workflow. Freshly isolated peripheral blood mononuclear cells (PBMCs) underwent red blood cell (RBC) lysis and CD3 enrichment. Samples were then subsequently stained with Hoechst/CD3/CD4/CD8 or Hoechst/CD3/CD8/RubyDead Dye surface marker kits and measured on the Cellaca PLX and three different flow cytometers for side-by-side comparison and assay validation. Acquisition and analysis of cell viability and cell populations was shown to be faster and more efficient process compared to flow while achieving highly comparable results between the two technology platforms. This data shows that the Cellaca PLX Image Cytometer may provide a rapid alternative or orthogonal method for PBMC immunophenotyping experiments, as well as potentially streamline the workflow to quickly move precious patient samples downstream within the development processes.

Multisite EGFR phosphorylation is regulated by adaptor protein abundances and dimer lifetimes.

Differential epidermal growth factor receptor (EGFR) phosphorylation is thought to couple receptor activation to distinct signaling pathways. However, the molecular mechanisms responsible for biased signaling are unresolved due to a lack of insight into the phosphorylation patterns of full-length EGFR. We extended a single-molecule pull-down technique previously used to study protein-protein interactions to allow for robust measurement of receptor phosphorylation. We found that EGFR is predominantly phosphorylated at multiple sites, yet phosphorylation at specific tyrosines is variable and only a subset of receptors share phosphorylation at the same site, even with saturating ligand concentrations. We found distinct populations of receptors as soon as 1 min after ligand stimulation, indicating early diversification of function. To understand this heterogeneity, we developed a mathematical model. The model predicted that variations in phosphorylation are dependent on the abundances of signaling partners, while phosphorylation levels are dependent on dimer lifetimes. The predictions were confirmed in studies of cell lines with different expression levels of signaling partners, and in experiments comparing low- and high-affinity ligands and oncogenic EGFR mutants. These results reveal how ligand-regulated receptor dimerization dynamics and adaptor protein concentrations play critical roles in EGFR signaling.

Crosstalk between immune cells and adipocytes requires both paracrine factors and cell contact to modify cytokine secretion.

Increased adiposity results in a heightened infiltration of immune cells into fat depots, which in turn generates a pro-inflammatory phenotype in obese individuals. To better understand the causal factors that establish this pro-inflammatory profile, we examined events leading to crosstalk between adipocytes and immune cells. Using isolated spleen-derived immune cells, stimulated with LPS, together with cultured adipocytes, we differentiated the effects of paracrine factors and cell-cell contact on TNFα, IL-6 and MCP-1 secretion levels and secretion profiles. When splenocytes and adipocytes were co-cultured without direct contact, permitting only paracrine communication, secretion of IL-6 and MCP-1 were increased by 3- and 2.5-fold, respectively, over what was secreted by individual cultures, whereas TNFα secretion was reduced by 55%. When cells were co-cultured with direct cell-cell contact, IL-6 and MCP-1 secretion were increased by an additional 36% and 38%, respectively, over that measured from just paracrine stimulation alone, indicating that cell contact provides a synergistic signal that amplifies elevated cytokine secretion stimulated by paracrine signals. Using splenocytes from TNFα(-/-) mice showed that the absence of TNFα has little effect on paracrine stimulation of cytokine secretion, but attenuates cell contact-mediated enhancement of IL-6 and MCP-1 secretion. Furthermore, TNFα supports cell contact-mediated signaling in part, but not exclusively, through Nuclear Factor-κB activation. These findings indicate that engagement of cell contact between immune cells and adipocytes, in conjunction with locally secreted paracrine factors, activates a unique signaling pathway that mediates crosstalk between these cell types leading to marked effects on cytokine secretion and profile.