A pipeline to evaluate inhibitors of the Pseudomonas aeruginosa exotoxin U.

Pseudomonas aeruginosa has recently been highlighted by the World Health Organisation (WHO) as a major threat with high priority for the development of new therapies. In severe P. aeruginosa infections, the phospholipase activity of the type 3 secretion system toxin, ExoU, induces lysis of target host cells and results in the poorest clinical outcomes. We have developed an integrated pipeline to evaluate small molecule inhibitors of ExoU in vitro and in cultured cell models, including a disease-relevant corneal epithelial (HCE-T) scratch and infection model using florescence microscopy and cell viability assays. Compounds Pseudolipasin A, compound A and compound B were effective in vitro inhibitors of ExoU and mitigated P. aeruginosa ExoU-dependent cytotoxicity after infection of HCE-T cells at concentrations as low as 0.5 µM. Addition of the antimicrobial moxifloxacin controlled bacterial load, allowing these assays to be extended from 6 h to 24 h. P. aeruginosa remained cytotoxic to HCE-T cells with moxifloxacin, present at the minimal inhibitory concentration for 24 h, but, when used in combination with either Pseudolipasin A, compound A or compound B, a greater amount of viable cells and scratch healing were observed. Thus, our pipeline provides evidence that ExoU inhibitors could be used in combination with certain antimicrobials as a novel means to treat infections due to ExoU producing P. aeruginosa, as well as the means to identify more potent ExoU inhibitors for future therapeutics.

Effect of intraocular surgery and ketamine on aqueous and serum cytokines.

PURPOSE: To determine the effect of intraocular surgery and anesthesia on aqueous and serum cytokines. METHODS: Patients undergoing routine cataract surgery under general and local (peribulbar) anesthesia were randomized to those given general anesthetic with and without the use of ketamine and those having local anesthesia. Aqueous and serum levels of cytokines were collected at commencement of surgery and were determined by an immunoassay using multi-analyte biochip array technology at 18 h post-operatively. RESULTS: At 18 h postoperative, all patients (37) showed significant and many fold increases in their aqueous levels of interleukin (IL)-1alpha, IL-1beta, IL-4, IL-6, IL-8, vascular endothelial growth factor (VEGF), tumor necrosis factor (TNF)-alpha, interferon (IFN)-gamma, epidermal growth factor (EGF), and monocyte chemotactic protein (MCP)-1. There was little to no increase in IL-2 and IL-10. Significant increases in some cytokines (EGF, IL-6, and IFN-gamma) in the serum were also found (p=0.038). There were no significant differences in aqueous cytokine levels following the use of ketamine or between those patients who had general and local anesthesia (0.11