The Pro-inflammatory Effects of LL-37 Antimicrobial Peptide on Induction of Arachidonic Acid Metabolism and Interleukin-8 Synthesis in Human Fibroblasts from Periodontal Tissue
NB: Please be notified for some changes in the main and specific objectives. This is due to much reduced amount of fund to support this research project. Therefore, I would like to down-size the project to study only one type of cells, i.e. human gingival fibroblasts, instead of both human gingival fibroblasts and human periodontal ligament fibroblasts. Furthermore, I will only focus on the effects of LL-37 antimicrobial peptide, rather than both epithelial-derived -defensins and LL-37, on arachidonic acid metabolism and IL-8 synthesis, since I currently collaborate with Professor Jan Bolscher, ACTA, the Netherlands, for free LL-37 peptide and the cost of epithelial-derived -defensins is way too high to be affordable by only this small budget.
1. To determine the pro-inflammatory effects of oral epithelial- and neutrophil-derived LL-37 antimicrobial peptide in human gingival fibroblasts.
1a) To determine the pro-inflammatory effects of LL-37 by analyses of COX-2 and IL-8 mRNA up-regulation in dose- and time-dependent manners.
1b) To determine the pro-inflammatory effects of LL-37 by analyses of COX-2 and IL-8 protein expression in dose- and time-dependent fashions.
1c) To determine the pro-inflammatory effects of LL-37 by analyses of PGE2 levels in conditioned media in dose- and time-dependent manners.
1d) To determine cell viability after incubation with LL-37.
1e) To determine whether elevated PGE2 levels in response to treatment with LL-37 are due to up-regulated COX-2 expression.
2. To investigate the signaling pathways involved in COX-2 and IL-8 up-regulation.
2a) To determine the phosphorylation of MAP kinase pathways after cell treatment with LL-37 for different times in whole cell lysates.
2b) To examine the activation of NF- B by nuclear translocation.
2c) To investigate the presence of specific NF- B isoforms in nuclear extract in comparison with whole cell extract of treated cells.
2d) To investigate the functional activity and the specific NF- B isoforms in nuclear extract of treated cells.
2e) To determine whether specific pharmacological inhibitors for MAP kinase or NF- B pathways can abrogate up-regulation of IL-8 and COX-2, and its respective product, i.e. PGE2.
3. To study the role of epidermal growth factor (EGF) and its receptor (EGFR) in regulating IL-8 or COX-2 expression. Alternatively, other receptors that were previously reported to be involved with cellular activation by LL-37, including purinergic receptors, will be studied.
3a) To examine expression of EGFR and purinergic receptors, particularly the P2X family, in human gingival fibroblasts.
3b) To determine involvement of EGF, EGFR, and purinergic receptors in COX-2 and IL-8 up-regulation by using several pharmacological inhibitors.
