Harriman, KatelynLindblad, William2025-07-172025-04-17https://dspace.husson.edu/handle/20.500.14298/311It is known that wound healing in the diabetic person is impaired and is characterized by excessive inflammation. However, the underlying mechanism(s) contributing to this heightened inflammatory response remain largely unknown. Previous studies from this laboratory, and others, have shown that fibroblasts can secrete pro-inflammatory mediators in response to exposure to lipopolysaccharide (LPS), a known inducer of inflammation. Further, fibroblasts obtained from type I diabetic individuals (GM01842) show significantly enhanced secretion of pro-inflammatory mediators compared to non-diabetic control cells (GM23973). This study had two primary aims, namely to investigate whether this increased secretion is due to elevated expression of CD14/TLR4 receptors, and whether LPS binding correlates with the expression of pro-inflammatory interleukin-8 (IL-8) versus extracellular matrix protein type I collagen (COL1A1). Non-transformed human dermal fibroblasts from the Coriell Cell Repository were maintained under standard culture conditions. Cell binding conditions were optimized (temperature, time, and ligand concentration) for binding Alexa488-labelled LPS, and flow cytometry was used to quantify LPS binding. mRNA levels for COL1A1 and IL-8 were determined by RT-PCR using Bio-Rad SYP qPCR kits. Binding of LPS to GM01872 was shown to be dose dependent with 500 ng providing a significant increase in cell fluorescence. Of note, exposure of GM23973 cells to this LPS content suppressed fluorescence suggesting that the environment of the CD14 receptor may be altered. Given prior results and from the current study, IL-8 expression in GM01872 cells was significantly higher than GM23973 and whereas expression of COL1A1 was at non-treated levels. These findings provide additional insights into the dysregulated inflammation observed in diabetic wound healing. CD14/TLR4 binding environments may be altered to facilitate higher levels of pro-inflammatory gene expression by cells that have experienced the hyperglycemic state of diabetes. These data offer new targets for therapeutic intervention.en-USPoster