Differential cytotoxicity of long-chain bases for human oral gingival epithelial keratinocytes, oral fibroblasts, and dendritic cells

Abstract

Long-chain bases are present in the oral cavity. Previously we determined that sphingosine, dihydrosphingosine, and phytosphingosine have potent antimicrobial activity against oral pathogens. Here, we determined the cytotoxicities of long-chain bases for oral cells, an important step in considering their potential as antimicrobial agents for oral infections. This information would clearly help in establishing prophylactic or therapeutic doses. To assess this, human oral gingival epithelial (GE) keratinocytes, oral gingival fibroblasts (GF), and dendritic cells (DC) were exposed to 10.0-640.0 µM long-chain bases and glycerol monolaurate (GML). The effects of long-chain bases on cell metabolism (conversion of resazurin to resorufin), membrane permeability (uptake of propridium iodide or SYTOX-Green), release of cellular contents (LDH), and cell morphology (confocal microscopy) were all determined. GE keratinocytes were more resistant to long-chain bases as compared to GF and DC, which were more susceptible. For DC, 0.2 to 10.0 µM long-chain bases and GML were not cytotoxic; 40.0 to 80.0 µM long-chain bases, but not GML, were cytotoxic; and 80.0 µM long-chain bases induced cellular damage and death in less than 20 minutes. The LD50 of long-chain bases for GE keratinocytes, GF, and DC were considerably higher than their minimal inhibitory concentrations for oral pathogens, a finding important to pursuing their future potential in treating periodontal and oral infections.

Predicting PD-L1 expression on human cancer cells using next-generation sequencing information in computational simulation models

“…At 24 hours, cells were pelleted by centrifugation (400 RCF, Eppendorf 5810R, Brinkmann Instruments, Inc., Westbury, NY) at 4°C for 5 minutes, fixed in 10.0% neutral buffered formalin at room temperature, and suspended in 1.0% BactoAgar (Becton, Dickinson, and Co., Sparks, MD)-1.25% gelatin (Electron Microscopy Sciences, Hatfield, PA). BactoAgar-gelatin punches were made and plugs containing cells were immobilized together in a 12-plug array using molten BactoAgar-gelatin [34] and processed for microtomy as recently described [35]. …”

“…Flow cytometry was performed as recently described [35] to confirm cell surface expression of PD-L1. Briefly, 0.5 ml containing 0.5 × 10 5 viable cells in their respective media were incubated without and with IFNγ (10 ng/ml) for 24 hours.…”

Cytotoxicity of HBD3 for dendritic cells, normal human epidermal keratinocytes, hTERT keratinocytes, and primary oral gingival epithelial keratinocytes in cell culture conditions

“…The identities of GE369 and GE373 keratinocytes were confirmed using flow cytometry as recently described (Poulsen et al, 2015). Keratinocytes resuspended in fresh media at 10 5 viable cells/ml were blocked with human IgG (Sigma, Saint Louis, MO) for 20 min, washed with stain buffer (BD Pharmingen, San Jose, CA), and resuspended in 100 μl stain buffer (BD Pharmingen, San Jose, CA) at 10 6 cells/ml before adding antibodies to surface markers CD24 (PE-Cy7-CD24; BD Pharmingen, San Jose, CA) and CD104 (FITC-CD104; BioLegend, San Diego, CA).…”

Characterizing how glycerol monolaurate (GML) affects human T cell signaling and function

“…At GML concentrations that are antimicrobial, GML does not result in cytotoxicity for human gingival epithelial keratinocytes, oral fibroblasts, and dendritic cells [141]. However GML incorporated into tampons and GML infused gels applied topically in the vagina result in decreased IL-8 in the vaginal environment [130,138], suggesting that GML has an immunomodulatory role.…”