VGX-750

An inflammatory response is a hallmark of necrosis evoked by bacterial pathogens. Vibrio vulnificus, VvpE, is an elastase that is responsible for tissue necrosis and inflammation; however, the molecular mechanism by which it regulates host cell death has not been characterized. In the present study, we investigate the cellular mechanism of VvpE with regard to host cell death and the inflammatory response of human intestinal epithelial (INT-407) cells. The recombinant protein (r)VvpE (50 pg/ml) caused cytotoxicity mainly via necrosis coupled with IL-1β production. The necrotic cell death induced by rVvpE is highly susceptible to the knockdown of annexin A (ANXA)2 and the sequestration of membrane cholesterol. We found that rVvpE induces the recruitment of NADPH oxidase 2 and neutrophil cytosolic factor 1 into membrane lipid rafts coupled with ANXA2 to facilitate the production of reactive oxygen species (ROS). The bacterial signaling of rVvpE through ROS production is uniquely mediated by the phosphorylation of redox-sensitive transcription factor NF-κB. The silencing of NF-κB inhibited IL-1β production during necrosis. rVvpE induced hypomethylation and region-specific transcriptional occupancy by NF-κB in the IL-1β promoter and has the ability to induce pyroptosis via NOD-, LRR-, and pyrin domain–containing 3 inflammasome. In a mouse model of V. vulnificus infection, the mutation of the vvpE gene from V. vulnificus negated the proinflammatory responses and maintained the physiological levels of the proliferation and migration of enterocytes. These results demonstrate that VvpE induces the hypomethylation of the IL-1β promoter and the transcriptional regulation of NF-κB through lipid raft–dependent ANXA2 recruitment and ROS signaling to promote IL-1β production in intestinal epithelial cells.

In a previous report in Clin. Chem., the authors described much higher international normalized ratios (INRs) with recombinant Neoplastin R (Roche Diagnostics) than with tissue-extract Neoplastin Plus (Roche Diagnostics) in patients initiating oral anticoagulant therapy.For patients on stable oral anticoagulation, they observed no significant differences in INRs between the thromboplastins.In line with this, this study investigated whether the authors' findings could be extrapolated to other recombinant thromboplastins.There were increasing differences in the INR between recombinant thromboplastins (Innovin, RecombiPlasTin, Neoplastin R) and tissue-extract thromboplastins (Hepato Quick, Thromborel S, Neoplastin Plus) with increasing INR for all of the tested thromboplastins.Data showed that for patients with INR values above the therapeutic interval, 1.5-fold increases in the INR may be observed more frequently for recombinant thromboplastins than for tissue-extract thromboplastins.Hence, it would be significant for clinicians to consider the thromboplastin source when making dosage adjustments for patients with high INRs.