Simplifying the Execution of HepatoPac MetID Experiments: Metabolite Profile and Intrinsic Clearance Comparisons.

The HepatoPac micropatterned coculture (MPCC) hepatocyte system has been shown to be an effective tool to investigate the qualitative human and preclinical species' metabolite profiles of new drug candidates. However, additional improvements to the overall study conditions and execution, layout, and human-donor count could be made. To that end, we have evaluated several ways to increase the amount of data one can generate per MPCC plate and how to more efficiently execute a MPCC study for the purpose of metabolite generation. Herein, we compare a set of compounds using single- and 10-donor pooled human MPCC hepatocytes. Intrinsic clearance and mean metabolic activities assessed by diverse enzyme markers were comparable between the single- and 10-donor pool. We have confirmed that the generated metabolite profiles were indistinguishable between the single- and 10-donor pool and also that rat MPCC can be performed at 400 µl media volume, which greatly simplifies study execution. Additional tips for successful study execution are also described. SIGNIFICANCE STATEMENT: When using the HepatoPac micropatterned coculture (MPCC) system, sometimes simple experimental condition variables or problematic plate designs can hamper productive study execution. We evaluated conditions to increase the amount of data one can generate per MPCC plate and, perhaps more importantly, execute that study more efficiently with less likelihood of error. We describe some of our key learnings, provide an examination of enzyme activity levels and clearance values, and provide some recommendations to simplify the execution of a HepatoPac experiment.

Granzyme B is expressed in mouse mast cells in vivo and in vitro and causes delayed cell death independent of perforin.

Mast cells respond to pathogens and allergens by secreting a vast array of preformed and newly synthesized mediators, including enzymes, vasoactive amines, lipid mediators, cytokines and chemokines, thereby affecting innate and adaptive immune responses and pathogenesis. Here, we present evidence that skin-, but not lung-associated primary mast cells as well as in vitro-differentiated bone marrow-derived mast cells (BMMC) express granzyme (gzm) B, but not gzmA or perforin (perf). GzmB is associated with cytoplasmic granules of BMMC and secreted after Fcepsilon-receptor-mediated activation. BMMC from wild type but not gzmB-deficient mice cause cell death in susceptible adherent target cells, indicating that the perf-independent cytotoxicity of BMMC is executed by gzmB. Furthermore, gzmB induces a disorganization of endothelial cell-cell contacts. The data suggest that activated mast cells contribute, via secreted gzmB, to cell death, increased vascular permeability, leukocyte extravasation and subsequent inflammatory processes in affected tissues.