D. O'Rourke

The Bacteroides fragilis transcriptome response to oxygen and H2O2: the role of OxyR and its effect on survival and virulence

The intestinal anaerobic symbiont, Bacteroides fragilis, is highly aerotolerant and resistant to H2O2. Analysis of the transcriptome showed that expression of 45% of the genome was significantly affected by oxidative stress. The gene expression patterns suggested that exposure to oxidative stress induced an acute response to rapidly minimize the immediate effects of reactive oxygen species, then upon extended exposure a broad metabolic response was induced. This metabolic response induced genes encoding enzymes that can supply reducing power for detoxification and restore energy‐generating capacity. An integral aspect of the metabolic response was downregulation of genes related to translation and biosynthesis which correlated with decreased growth and entry into a stationary phase‐like growth state. Examination of oxyR mutants showed that they were impaired for the acute response and they induced the expanded metabolic response with only minimal exposure to stress. The oxyR mutants were more sensitive to oxidants in vitro and in vivo they were attenuated in an intra‐abdominal abscess infection model. Aerotolerance and resistance to oxidative stress are physiological adaptations of B. fragilis to its environment that enhance survival in extra‐intestinal sites and promote opportunistic infections.

Pilot studies of pressure-immobilization bandages for rattlesnake envenomations

Study objective. Pressure-immobilization bandages sequester venom in extremities and are recommended for snakebites without local toxicity. Pilot studies were performed to determine the time of onset of toxicity and efficacy of pressure-immobilizations bandages in a porcine model of rattlesnake envenomation. Methods. After IACUC approval, anesthetized pigs were injected subcutaneously in a distal hind leg with 200 mg of Crotalus atrox venom. After 1 min, pigs received either a pressure-immobilization bandage (N = 3) or no treatment (N = 3). At 24 h, surviving pigs received antivenin and then the pressure-immobilization bandages were removed. Surviving subjects were followed for 1 week. Chi-square analysis and paired t-test were used. Results. Pigs with pressure-immobilization bandages survived for 24 h, whereas untreated pigs died at 13.68 ± 3.42 h (p = 0.014). Surviving pigs walked on the extremity at 7 days. Potassium rose from 4.033 ± 0.252 at baseline to 17.767 ± 5.218 mEq/L (p < 0.0001) at time of death in untreated pigs but was normal at 24 h in treated subjects. Widespread tissue necrosis was seen in the untreated group but only local necrosis in the treatment group. Conclusions. Pressure-immobilization bandages prevented death from severe C. atrox envenomations with a 24 h delay to treatment. Surviving pigs had recovery of limb use at 1 week.

A Localizing Circumferential Compression Device Increases Survival after Coral Snake Envenomation to the Torso of an Animal Model

BACKGROUND Pressure immobilization bandages have been shown to delay onset of systemic toxicity after Eastern coral snake (Micrurus fulvius) envenomation to the distal extremity. OBJECTIVES To assess the efficacy of a novel compression device in delaying onset of systemic toxicity after truncal envenomations with Eastern coral snake (Micrurus fulvius) venom in a porcine model. METHODS With University approval, nine juvenile pigs (11 kg to 22 kg) were sedated, anesthetized, and intubated but not paralyzed to ensure continuous spontaneous respirations in a university animal laboratory. Each animal was injected subcutaneously with 10 mg of M. fulvius venom in a pre-selected area of the trunk. After 1 min, six animals had the application of a novel, localizing circumferential compression (LoCC) device applied to the bite site (treatment group) and three animals had no treatment (control group). The device was composed of a rigid polymer clay form molded into a hollow fusiform shape with an internal dimension of 8 × 5 × 3 cm and an elastic belt wrapped around the animal securing the device in place. Vital signs were recorded at 30-min intervals. End points included a respiratory rate below 3 breaths/min, oxygen saturation < 80%, or survival to 8 h. Survival to 8 h was analyzed using Fishers exact test, with p < 0.05 indicating significance. Survival analysis was performed using the Mantel-Cox test to assess time to death with outcomes represented in a Kaplan-Meier Cumulative survival plot. RESULTS Five of the six pigs in the treatment group survived 8 h (293-480 min). None of the control pigs survived to 8 h (Fishers exact p = 0.04), with mean time of respiratory failure 322 min (272-382 min). Survival analysis showed a significant delay in time to event in the treatment group compared to the control group (p = 0.04). CONCLUSIONS The LoCC device used in this study delayed the onset of systemic toxicity and significantly increased survival time after artificial truncal envenomation by Eastern coral snake venom.

GPR4 Deficiency Alleviates Intestinal Inflammation in a Mouse Model of Inflammatory Bowel Disease

GPR4 is a proton-sensing G protein-coupled receptor that can be activated by extracellular acidosis. It has recently been demonstrated that activation of GPR4 by acidosis increases the expression of numerous inflammatory and stress response genes in vascular endothelial cells (ECs) and also augments EC-leukocyte adhesion. Inhibition of GPR4 by siRNA or small molecule inhibitors reduces endothelial cell inflammation. As acidotic tissue microenvironments exist in many types of inflammatory disorders, including inflammatory bowel disease (IBD), we examined the role of GPR4 in IBD using a dextran sulfate sodium (DSS)-induced colitis mouse model. We observed that GPR4 mRNA expression was increased in mouse and human IBD tissues when compared to control intestinal tissues. To determine the function of GPR4 in IBD, wild-type and GPR4-deficient mice were treated with 3% DSS for 7 days to induce acute colitis. Our results showed that the severity of colitis was decreased in GPR4-deficient DSS-treated mice in comparison to wild-type DSS-treated mice. Clinical parameters, macroscopic disease indicators, and histopathological features were less severe in the DSS-treated GPR4-deficient mice than the DSS-treated wild-type mice. Inflammatory gene expression, leukocyte infiltration, and isolated lymphoid follicle (ILF) formation were reduced in intestinal tissues of DSS-treated GPR4-null mice. Collectively, our results suggest GPR4 provides a pro-inflammatory role in IBD as the absence of GPR4 ameliorates intestinal inflammation in the acute DSS-induced IBD mouse model.